Several years before Jonas Salk and Albert Sabin developed their famous polio vaccines, Hilary Koprowski (1916-2013) in fact developed the world’s first effective, but much less well known polio vaccine (1, 2). Koprowski’s vaccine was used world-wide, but it was never licensed in the United States, ultimately losing out to Sabin’s vaccine.

Koprowski’s reputation was tarnished in 1950, when he tested his live polio vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY; an episode recounted in a recent posting Vaccine Research Using Children (1). Koprowski reported on the Letchworth Village trials at a 1951 conference of major polio researchers. Although his vaccine induced immunity in the children, and caused no ill effects, many scientists in the audience were horrified that he actually tested a live polio vaccine in human children. Afterwards, Sabin shouted at him: “Why did you do it? Why? Why?”

Although Koprowski’s polio vaccine was supplanted by the Salk and Sabin vaccines, his demonstration, that a live polio vaccine could be safe and effective, paved the way for Sabin to develop his live polio vaccine. Moreover, Sabin developed his vaccine from a sample of attenuated poliovirus that he received from Koprowski.

There is much more to tell about Koprowski. This posting relates some of the remarkable earlier events of his life, including his harrowing escape from Poland on the eve of the Second World War; a flight which inadvertently led to his career in virology. A subsequent posting will recount the now discredited, although sensational at the time, accusation that Koprowski’s polio vaccine gave rise to the HIV/AIDS epidemic.

Koprowski was born and grew up in Warsaw, where he earned a medical degree from Warsaw University in 1939. He also was an accomplished pianist, having studied piano from the age of 12 at the prestigious Warsaw Conservatory, where Chopin is said to have studied. Koprowski eventually earned a music degree from the Conservatory. He recalled, “…the first year I was the youngest and voted second best in the class (3).”

Hilary Koprowski in Warsaw (2007)

In 1938, while Koprowski was in medical school, he married classmate Irena Grasberg who, in later years, would wonder how they had found the time for their courtship. Each had to contend with a demanding medical school program, while Hilary’s piano studies at the Conservatory was a full time program in itself (3). Irena recalled a day before both of them had an anatomy exam, and Hilary had an important recital. Hilary practiced a recital piece, while simultaneously studying a chart on the music rack showing the bones of the hand; all the while as Irena read anatomy to him.

Koprowski eventually chose a career in medicine, rather than one in music. As he explained: “…the top of the music pyramid is much narrower than that of medicine, where there is more space for successful scientists (3).” Koprowski rated himself only fourth best in his class at the Warsaw Conservatory, and he needed to excel. Yet he may have underrated himself. His piano professor at the Conservatory was “greatly disappointed” when he chose to enter medicine (3). [After the 1944 Warsaw uprising, Koprowski’s piano professor was arrested and beaten to death by German soldiers (see below and 3).] In any case, Koprowski continued to play the piano, and he even did some composing in his later years.

Germany invaded Poland in September 1939, setting off the Second World War. As German bombs were falling on Warsaw, Koprowski answered the call for Polish men to go east, where Polish forces were organizing to resist the Germans. Irena, now pregnant, and Hilary’s mother went with him, while his father chose to remain behind. They made their way in a horse-drawn hay wagon, traveling at night to avoid German planes that were strafing the roads during the day. After a week or so on the road, they encountered refugees moving in the opposite direction. Those refugees told them that Russia had signed a pact with Germany and was now invading Poland from the east (Aside 1). So the three Koprowskis joined the flood of refugees moving to the east. When they arrived back in Warsaw, they found the city in ruins. Many of their friends and neighbors had been killed or were seriously wounded, and the city was occupied by German soldiers.

[Aside 1: The German–Soviet Non-aggression Pact was signed in Moscow in August 1939, as a guarantee of non-belligerence between Nazi Germany and the communist Soviet Union. Hitler broke the pact in June 1941 when Germany attacked Soviet positions in eastern Poland. Hitler had no intention of keeping to the pact. However, it temporarily enabled him to avoid having to fight a war on two fronts—against Britain and France in the west and the Soviet Union in the east.]

Once Germany had conquered Poland, German and Polish Jews began to be sent to concentration camps set up in Poland. The Koprowskis, who were Jewish (Salk and Sabin too were descendants of eastern European Jews), quickly made plans to leave Poland. Their first destination was to be Rome. Hilary’s father went there first to arrange living conditions for the family. To facilitate the escape of Hilary’s father from Poland, Hilary and Irena wrapped him in bandages, hoping that the authorities might gladly believe they were letting a very frail individual depart from the country.

Hilary, Irena, and Hilary’s mother then traveled by train from Warsaw to Rome. It was a harrowing trip. Irena was pregnant, and the Gestapo was roaming the trains. They feared that they might have been arrested at any time.

In Rome, the Koprowski family’s main concern was the safety of Irena and her unborn baby. Since Irena had an aunt in Paris, who would know of a good doctor there, the family thought that Paris would be a safe place for the baby to be born. Thus, Irena left for Paris, accompanied by Hilary’s father. She gave birth to Claude five days after arriving there.

Hilary did not go with Irena to France. If he had done so, he would have been impressed immediately into the Polish Army that was forming there to fight the Germans. Yet he knew that he would eventually have to leave Rome. Italy, under Mussolini’s leadership, was poised to enter the Second World War, as an Axis partner of Hitler’s Germany.

After Claude was born, Irena worked as a physician at a psychiatric hospital in Villejuif, just outside of Paris. She was the sole internist there for eight hundred patients. She kept Claude at the hospital, in a locked room, which she would slip to away every three hours to nurse him.

Back in Rome, Hilary continued to play the piano. In fact, he auditioned for, and was accepted by Rome’s L’Accademia di Santa Cecilia, which awarded him a second degree in music. Importantly, his skill at the keyboard enabled him to get visas for himself and his mother to enter Brazil, which the family hoped would be a safe haven. The best students from L’Accademia di Santa Cecilia were often in demand to play for events at the Brazilian embassy in Rome. Thus, on several occasions, Hilary played the piano at the embassy. Brazil’s consul general admired Hilary’s pianism and was pleased to arrange Brazilian entry visas for Hilary and his mother. See Aside 2.

[Aside 2: The day after Hilary arrived in Rome, he volunteered to serve as a medical examiner for a Polish draft board that was set up in the Polish embassy. The draft board’s activity at the embassy—recruiting Poles for the Polish Army—violated diplomatic protocol. In addition, Italy would soon be Germany’s Axis partner in the War. Moreover, Brazil, though neutral in the War, favored the Axis.]

Hilary and his mother had been making plans to leave Italy. Their destination was to be Spain, where they hoped they might unite with Irena, Claude, and Hilary’s father. From Spain, the family might then go to Portugal, where they could get a boat to Brazil. But, on the very day that Hilary and his mother were to leave Italy, Mussolini issued a proclamation banning any male of military age from leaving the country. So it happened that Hilary’s escape from Italy was blocked at the boat registration. However, his mother rose to the occasion, crying and pleading with the boat registration official that she was sick, that Hilary was her sole means of support, and that she could not go on without him. “The man looked at his watch and said he must go to lunch. He looked at us and said, ‘If the boat leaves before I return, that’s my bad luck (3).’” So, Hilary and his mother boarded the boat, which left before the official returned. [Hilary’s mother was a well-educated woman, and a dentist by profession.]

In Spain, Hilary and his mother stayed at a hotel in Barcelona. Despite the wartime conditions, they were able to communicate, if only sporadically, with Irena and Hilary’s father, who were still in France. Then, after Germany invaded France in 1940, Irena, Claude, and Hilary’s father reunited with Hilary and his mother in Barcelona. [The escape of Irena, Claude, and Hilary’s father from France was far more harrowing than the escape of Hilary and his mother from Italy (See 3 for details).]

The family now needed to get to Portugal, where they could then get a boat to Brazil. Irena had already obtained Portuguese visas for herself and for Claude. But Hilary and his mother only had visas for Brazil. Hilary’s applications for visas at the Portuguese embassy were repeatedly denied, until a fellow Pole at Hilary’s Barcelona hotel advised him of the obligatory bribe that must accompany visa applications. The advice was right-on, and the family (minus Hilary’s father, who chose to go to England) sailed for Brazil without further incident.

In Brazil, Irena found work in Rio de Janeiro as a nurse. But she soon managed to secure a position as a pathologist at the largest hospital in the city. Hilary, on the other hand, could not find a job in medicine and, so, he turned to teaching piano. After six months of teaching unenthusiastic piano students, Hilary by chance recognized a man on the street in Rio who happened to be a former schoolmate from Warsaw. The man also happened to be working at the Rockefeller Foundation’s outpost in Rio. He told Hilary that the Foundation was looking for people, and he also told Hilary who he should contact there. Hilary interviewed at the Foundation the next day, and was told to report for work the day after that.

The Foundation assigned Hilary to research how well, and for how long the attenuated yellow fever vaccine—developed by Nobel laureate Max Theiler in 1935 (4) —might protect against yellow fever. The disease was endemic in Brazil, and it was actually the Rockefeller Foundation’s first priority.

Hilary’s supervisor at the Foundation was Edwin Lennette; a staff member of the International Health Division of the Rockefeller Foundation, assigned to its Brazilian outpost, specifically because of his interest in yellow fever. In 1944, Lennette would be reassigned to the Rockefeller Foundation laboratory in Berkeley, California, where he would establish the first diagnostic virology laboratory in the United States. Indeed, Lennette is known as one of the founders of diagnostic virology. But, in Brazil, he introduced Hilary Koprowski to virology.

Hilary’s apprenticeship under Lennette was going very well. It would result in nine papers—published between 1944 and 1946— that Hilary would co-author with Lennette. Moreover, Lennette was interested in other viruses, in addition to yellow fever. Thus, their co-authored papers included studies of Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, and West Nile virus, as well as yellow fever.

Most importantly, Koprowski’s work under Lennette introduced him to Max Theiler’s methods and approach to viral attenuation. In brief, Theiler found that propagating yellow fever virus in an unnatural host—chick embryos—caused the virus to adapt to that host, thereby reducing its capacity to cause disease in humans. Koprowski would later acknowledge that Theiler provided him with a “most encouraging model” for attenuating poliovirus. [Koprowski attenuated poliovirus by propagating it first in mice and then in rats. Recall that Sabin developed his live polio vaccine from attenuated poliovirus that he received from Koprowski (1).] See Asides 3 and 4.

[Aside 3: The rabies vaccine, which Louis Pasteur developed in 1885, is often referred to as the first attenuated virus vaccine. Nevertheless, while Pasteur did passage his vaccine virus in rabbit spinal cords, the virus may have been killed when the spinal cords were later dried for up to fourteen days. Also, in Pasteur’s day, nothing was known about immunity or mutation, and viruses had not yet been identified as microbes distinct from bacteria. The yellow fever vaccine developed by Max Theiler at the Rockefeller Institute (now University) in New York may have been the first deliberately attenuated viral vaccine.]

[Aside 4: Koprowski and Lennette were among the first researchers to observe that infection by one virus (yellow fever, in this instance) might inhibit the growth of another unrelated virus (West Nile virus, in this instance). That is, they had inadvertently detected what later would be known as interferon. Yet while they looked for an anti-viral substance in their tissue culture media, and while their results suggest that it actually was there, they stated in their summary that nonspecific anti-viral factors were not present (5). Koprowski and Lennette collaborated again in the 1970s; this time to investigate subacute sclerosing panencephalitis, a rare late complication of measles infection that results in neurodegeneration.]

Hilary continued to give piano recitals in Brazil, regretting only that he did not have time to practice the piano as much as he would have liked. Nonetheless, his piano playing expanded his circle of friends to include musicians, artists and writers, in addition to his fellow scientists. Moreover, Irena was satisfied with her medical practice, and with the many friends and rich social life that she and Hilary had in Brazil.

Earlier, in 1940, while Hilary was still in Rome, and expecting that the family would soon have to leave Europe, he believed that the United States would likely be the best destination for them. Thus, he applied to the United States for visas. He had nearly forgotten those applications when, in 1944, their numbers came up.

The Koprowski family now faced somewhat of a dilemma. It was happily settled in Brazil, and had no prospects in the United States. On the other hand, the Rockefeller Foundation’s yellow fever project was drawing to a close, and the Foundation was planning to leave Rio. Importantly, coming to America was now a “dream come true (3)”. So, in December 1944, the Koprowskis boarded an aging steamer in Brazil, and sailed under wartime blackout conditions, through German submarine-infested waters, for New York City.

During Hilary’s his first days in America, he used the Rockefeller Institute library in Manhattan to work on manuscripts reporting his research in Brazil. During one of his visits to the Rockefeller, he happened to meet Peter Olitzky (Aside 5), an early polio researcher there, who arranged for Hilary to meet Harold Cox, the director of the virology department at Lederle Laboratories, in Pearl River, New York. Hilary interviewed with Cox, who offered him a research position at Lederle, which Hilary accepted. Meanwhile, Irena was appointed an assistant pathologist at Cornell Medical College in Manhattan.

[Aside 5: In 1936, Olitzky and Sabin collaborated on a study at the Rockefeller Institute, which, although carefully done, wrongly concluded that poliovirus could attack nerve cells only; a result that did not bode well for the development of an attenuated polio vaccine.]

At Lederle, Hilary began the experiments that led to the world’s first successful polio vaccine. In 1950 he tested the live vaccine in eighteen mentally disabled children at Letchworth Village (1). None of these children had antibodies against poliovirus before he vaccinated them, but each of them was producing poliovirus antibodies after receiving the vaccine. Importantly, none of the children suffered ill effects. What’s more, Koprowski did not initiate the test. Rather, a Letchworth Village physician, fearing an outbreak of polio at the facility, came to Koprowski’s office at Lederle, requesting that Koprowski vaccinate the Letchworth children (1).

References:

Vaccine Research Using Children, Posted on the blog July 7, 2016.

Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.

Roger Vaughan, Listen to the Music: The Life of Hilary Koprowski. Springer-Verlag, 2000.

The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 4014.

Children have been used in vaccine research since its very beginning, usually said to have been in 1796, when Edward Jenner inoculated 8-year-old James Phipps with cowpox, and then challenged young James with actual smallpox (1). However, earlier, in 1789, Jenner inoculated his own 10-month-old son, Edward Jr., with swinepox. Edward Jr. then came down with a pox disease, which he fortunately recovered from. His father then challenged him with smallpox.

Edward Jr. survived his exposure to smallpox. But, since Edward Sr. wanted to determine the duration of young Edward’s protection, he again challenged his son with smallpox in 1791, when the boy was two. Edward Sr. inoculated his son yet again with smallpox when the boy was three. Fortunately, young Edward was resistant to each of the smallpox challenges his father subjected him to.

Jenner used several other young children in his experiments, including his second son, Robert, who was 11-months-old at the time. One of the children in Jenner’s experiments died from a fever; possibly caused by a microbial contaminant in an inoculum. [Microbes were not known in the late 18th century.]

We have no record of how Jenner (or his wife) felt about his use of his own children. However, there is reason to believe that Jenner felt some remorse over his use of James Phipps, who he referred to as “poor James.” Jenner looked after Phipps in later years, eventually building a cottage for him; even planting flowers in front of it himself.

By the 20th century, some of the most esteemed medical researchers were using children—in institutions for the mentally deficient—to test new drugs, vaccines, and even surgical procedures. These institutions were typically underfunded and understaffed. Several of them were cited for neglecting and abusing their residents. Moreover, their young patients were usually from poor families, or were orphans, or were abandoned. Thus, many of the children had no one to look out for their interests. In addition, research at these institutions was hidden from the public. [The goings-on at these institutions were, in general, hidden from the public, and most of the public likely preferred it that way.] Federal regulations that might have protected the children were not yet in existence, and federal approval was not even required to test vaccines and drugs.

In the early 1940s, Werner Henle, of the University of Pennsylvania, used children at Pennhurst—a Pennsylvania facility for the mentally deficient—in his research to develop an influenza vaccine. [Pennhurst was eventually infamous for its inadequate staffing, and for neglecting and abusing its patients (2). It was closed in 1987, after two decades of federal legal actions.] Henle would inoculate his subjects with the vaccine, and then expose them to influenza, using an oxygen mask fitted to their faces.

Pennhurst, a state-funded Pennsylvania facility for the mentally deficient, was one of the most shameful examples of the neglect and mistreatment that was common at these institutions. It was the site of Werner Henle’s research in the 1940s to develop an influenza vaccine.

Henle’s vaccine did not protect all of his subjects. Moreover, it frequently caused side effects. Additionally, Henle maintained (correctly?) that a proper test of a vaccine must include a control group (i.e., a group exposed to the virus, but not to the vaccine). Thus, he deliberately exposed unvaccinated children to influenza. Children who contracted influenza had fevers as high as 104o F, as well as typical flu-like aches and pains.

Despite Henle’s investigations at Pennhuerst, he was a highly renowned virologist, best known for his later research on Epstein Barr virus. See Aside 1.

[Aside 1: While Henle was researching his influenza vaccine at Pennhurst, Jonas Salk concurrently worked on an influenza vaccine, using adult residents (ranging in age from 20 to 70 years) at the Ypsilanti State School in Michigan.]

Next, consider Hilary Koprowski, an early competitor of Jonas Salk and Albert Sabin in the race to develop a polio vaccine (3). By 1950, Koprowski was ready to test his live polio vaccine in people. [That was four years before Sabin would be ready to do the same with his live polio vaccine.] Koprowski had already found that his vaccine protected chimpanzees against polio virus. And, he also tested his vaccine on himself. Since neither he nor the chimpanzees suffered any ill effects, Koprowski proceeded to test his vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY. [Like Pennhurst, Letchworth Village too was cited for inadequately caring for its residents.] Seventeen of Koprowski’s inoculated children developed antibodies to the virus, and none developed complications.

Koprowski did not initiate his association with Letchworth. Actually, Letchworth administrators, fearing an outbreak of polio at the facility, approached Koprowski, requesting that he vaccinate the children. Koprowski gave each child “a tablespoon of infectious material” in half a glass of chocolate milk (4). Koprowski never deliberately infected the Letchworth children with virulent virus.

Koprowski reported the results of his Letchworth studies at a 1951 conference of major polio researchers, attended by both Salk and Sabin. When Koprowski announced that he actually had tested a live vaccine in children, many conferees were stunned, even horrified. Sabin shouted out: “Why did you do it? Why? Why (4)?” See Aside 2.

[Aside 2: In the 1930s, Canadian scientist Maurice Brodie tested a killed polio vaccine in twelve children, who supposedly had been “volunteered by their parents (4).” For a short time Brodie was hailed as a hero. However, too little was known at the time for Brodie to ensure that his formaldehyde treatment had sufficiently inactivated the live polio virus. Consequently, Brodie’s vaccine actually caused polio in several of the children. After this incident, most polio researchers could not conceive of ever again testing a polio vaccine, much less a live one, in children.]

Neither Koprowski nor Letchworth Village administrators notified New York State officials about the tests. Approval from the state would seem to have been required, since Koprowski later admitted that he was certain he would have been turned down. And, it is not clear whether Koprowski or the school ever got consent from the parents to use their children. However, recall there were not yet any federal regulations that required them to do so.

Koprowski was untroubled by the uproar over his use of the Letchworth children, arguing that his experiments were necessary. Yet he later acknowledged: “if we did such a thing now we’d be put on jail…” But, he added, “If Jenner or Pasteur or Theiler (see Aside 2) or myself had to repeat and test our discoveries [today], there would be no smallpox vaccine, no rabies vaccine, no yellow fever vaccine, and no live oral polio vaccine.” Moreover, he maintained that, secret or not, his use of the Letchworth children fit well within the boundaries of accepted scientific practice.

[Aside 2: Nobel laureate Max Theiler developed a vaccine against yellow fever in 1937; the first successful live vaccine of any kind (5). Theiler formulated a test for the efficacy of his vaccine, which did not involve exposing humans to virulent virus. Sera from vaccinated human subjects were injected into mice, which were then challenged with the Yellow Fever virus.]

Koprowski referred to the Letchworth children as “volunteers (6).” This prompted the British journal The Lancet to write: “One of the reasons for the richness of the English language is that the meaning of some words is continually changing. Such a word is “volunteer.” We may yet read in a scientific journal that an experiment was carried out with twenty volunteer mice, and that twenty other mice volunteered as controls.” See Aside 3.

[Aside 3: Koprowski was a relatively unknown scientist when he carried out his polio research at Letchworth. He later became a renowned virologist, having overseen the development of a rabies vaccine that is still used today, and having pioneered the use of therapeutic monoclonal antibodies. Yet, he is best remembered for developing the world’s first effective polio vaccine; several years before Salk and Sabin brought out their vaccines.

Most readers of the blog are aware that the Salk and Sabin vaccines are credited with having made the world virtually polio-free. What then became of Koprowski’s vaccine? Although it was used on four continents, it was never licensed in the United States. A small field trial of Koprowski’s vaccine in 1956, in Belfast, showed that its attenuated virus could revert to a virulent form after inoculation into humans. Yet a 1958 test, in nearly a quarter million people in the Belgian Congo, showed that the vaccine was safe and effective. Regardless, the vaccine’s fate was sealed in 1960, when the U.S. Surgeon General rejected it on safety grounds, while approving the safer Sabin vaccine. Personalities and politics may well have played a role in that decision (3, 4).

Interestingly, Sabin developed his vaccine from a partially attenuated polio virus stock that he received from Koprowski. It happened as follows. In the early 1950s, when Koprowski’s polio research was further along than Sabin’s, Sabin approached Koprowski with the suggestion that they might exchange virus samples. Koprowski generously sent Sabin his samples, but Sabin never reciprocated.

Koprowski liked to say: “I introduce myself as the developer of the Sabin poliomyelitis vaccine (7).” He and Sabin had a sometimes heated adversarial relationship during the time when their vaccines were in competition. But they later became friends.]

Sabin was at last ready to test his polio vaccine in people during the winter of 1954-1955. Thirty adult prisoners, at a federal prison in Chillicothe, Ohio, were the subjects for that first test in humans. [The use of prisoners also raises ethical concerns.]

Recall Sabin’s public outcry in 1951 when Koprowski announced that he used institutionalized children to test his polio vaccine. In 1954, Sabin sought permission to do the very same himself; asserting to New York state officials: “Mentally defective children, who are under constant observation in an institution over long periods of time, offer the best opportunity for the careful and prolonged follow-up studies…”

Although Sabin had already tested his attenuated viruses in adult humans (prisoners), as well as in monkeys and chimpanzees, the National Foundation for Infantile Paralysis, which funded polio research in the pre-NIH days of the 1950s, blocked his proposal to use institutionalized children. Thus, Sabin again used adult prisoners at the federal prison in Ohio. With the concurrence of prison officials, virtually every inmate over 21 years-old “volunteered,” in exchange for $25 each, and a possible reduction in sentence. None of the prisoners in the study became ill, while all developed antibodies against polio virus.

Testing in children was still a necessary step before a polio vaccine could be administered to children on a widespread basis. But, Sabin’s vaccine could not be tested in children in the United States. Millions of American children had already received the killed Salk vaccine, and the National Foundation for Infantile Paralysis was not about to support another massive field trial of a vaccine, in children, in the United States (3).

Then, in 1959, after a succession of improbable events, 10 million children in the Soviet Union were vaccinated with Sabin’s vaccine (3). The Soviets were so pleased with the results of that massive trial that they next vaccinated all seventy-seven million Soviet citizens under 20 years-of-age with the Sabin vaccine. That figure vastly exceeded the number of individuals in the United States, who were vaccinated with the rival Salk vaccine during its field trials.

Next up, we have Nobel laureate John Enders who, in the 1950’s, oversaw the development of the first measles vaccine. Enders and co-workers carried out several trials of their attenuated measles vaccine; first in monkeys and then in themselves. Since the vaccine induced an increase in measles antibody titers, while causing no ill effects, they next tested it in severely handicapped children at the Walter E. Fernald State School near Waltham, Massachusetts.

Enders seemed somewhat more sensitive than either Henle or Koprowski to the ethics of using institutionalized children. Samuel L. Katz, the physician on Enders’ team, personally explained the trial to every Fernald parent, and no child was given the vaccine without written parental consent. [Federal guidelines requiring that step still did not exist.] Also, no child was deliberately infected with virulent measles virus.

Katz personally examined each of the inoculated Fernald children every day. None of these children produced measles virus, while all of them developed elevated levels of anti-measles antibodies. Also, the Fernald School had been experiencing severe measles outbreaks before the Enders team vaccinated any of its children. But, when the next measles outbreak struck the school, all of the vaccinated children were totally protected.

In 1963, the Enders vaccine became the first measles vaccine to be licensed in the United States. Several years later it was further attenuated by Maurice Hilleman (8) and colleagues at Merck. In 1971, it was incorporated into the Merck MMR (measles, mumps, and rubella) vaccine. See Aside 4.

[Aside 4: Before Enders carried out his measles investigations he pioneered the growth of viruses in tissue culture. In 1949, Enders, and collaborators Thomas Weller and Frederick Robbins, showed that poliovirus could be cultivated in the laboratory. This development was crucial, allowing Salk and Sabin to grow a virtually unlimited amount of polio virus and, consequently, to develop their polio vaccines. In 1954, Enders, Weller, and Robbins were awarded the Nobel Prize for Physiology or Medicine for their polio virus work.]

It may surprise some readers that before the mid 1960s the so-called Nuremburg Code of 1947 comprised the only internationally recognized ethical guidelines for experimentation on human subjects. The Nuremburg Code was drawn up by an American military tribunal during the trial of 23 Nazi physicians and scientists for atrocities they committed while carrying out so-called “medical” experiments during World War II. [Sixteen of the 23 Nazis on trial at Nuremburg were convicted, and 7 of these were executed (see Note 1)].

The Nuremberg Code’s Directives for Human Experimentation contained strongly stated guidelines. Its tenets included the need to obtain informed consent (interpreted by some to prohibit research using children), the need to minimize the risks to human subjects, and the need to insure that any risks are offset by potential benefits to society.

But, despite the well-articulated principles of the Nuremberg Code, it had little effect on research conduct in the United States. Federal rules, with the authority to regulate research conduct, would be needed for that. So, how did our current federal oversight of research come to be?

A 1996 paper in the The New England Journal of Medicine, “Ethics and Clinical Research,” by physician Henry Beecher, brought to the fore the need for rules to protect human subjects in biomedical research (9). Beecher was roused to write the paper in part by the early 1960s experiments of Saul Krugman, an infectious disease expert at NYU. Krugman used mentally deficient children at the Willowbrook State School in Staten Island, New York, to show that hepatitis A and hepatitis B are distinct diseases (9). Also, before a hepatitis vaccine was available, Krugman inoculated the children with serum from convalescing individuals, to ask whether that serum might protect the children against hepatitis. Krugman exposed the children to live virus either by injection, or via milkshakes seeded with feces from children with hepatitis.

Krugman found that convalescent sera indeed conferred passive immunity to hepatitis. Next, he discovered that by infecting passively protected patients with live hepatitis virus he could produce active immunity. Krugman had, in fact, developed the world’s first vaccine against hepatitis B virus (HBV) (see Aside 4). [Although Krugman used mentally deficient institutionalized children in his experiments, his investigations were nonetheless funded in part by a federal agency; the Armed Forces Epidemiology Section of the U.S. Surgeon General’s Office.]

[Aside 4: The first hepatitis B vaccine licensed for widespread use was developed at Merck, based on principles put forward by Nobel Laureate Baruch Blumberg, (10).]

Beecher was particularly troubled by two aspects of Krugman’s experiments. First, Krugman infected healthy children with live virulent virus. Beecher maintained that it is morally unacceptable to deliberately infect any individual with an infectious agent, irrespective of the potential benefits to society. [See reference 11 for an alternative view. “The ethical issue is the harm done by the infection, not the mere fact of infection itself.”]

Second, Beecher charged that the Willowbrook School’s administrators coerced parents into allowing their children to be used in Krugman’s research. The circumstances were as follows. Because of overcrowding at the school, Willowbrook administrators closed admission via the usual route. However, space was still available in a separate hepatitis research building, thereby enabling admission of additional children who might be used in the research.

Were the Willowbrook parents coerced into allowing their children to be used in the research there? Consider that the parents were poor and in desperate need of a means of providing care for their mentally impaired children. Making admission of the children contingent on allowing them to be used in the research might well be viewed as coercion. Yet even today, with federal guidelines now in place to protect human subjects, institutions such as the NIH Clinical Center admit patients who agree to participate in research programs. Is that coercion?

Beecher’s 1966 paper cited a total of 22 instances of medical research that Beecher claimed were unethical (9). Four examples involved research using children. Krugman’s work at Willowbrook was the only one of these four examples that involved vaccine research. Beecher’s other examples involved research using pregnant women, fetuses, and prisoners. But it was Beecher’s condemnation of Krugman’s hepatitis research at Willowbrook that is mainly credited with stirring debate over the ethics of using children in research.

Did Krugman deserve Beecher’s condemnation? Before Krugman began his investigations at Willowbrook, he plainly laid out his intentions in a 1958 paper in the New England Journal of Medicine (12). Importantly, Krugman listed a number of ethical considerations, which show that he did not undertake his Willowbrook investigations lightly. In fact, Krugman’s ethical considerations, together with his plans to minimize risks to the children, were not unlike the assurances one might now submit to an institutional review board (11).

Many (but not all) knowledgeable biomedical researchers claimed that Beecher misunderstood Krugman’s research and, thus, unjustly vilified him. Krugman was never officially censored for his Willowbrook investigations. Moreover, condemnation of Krugman did not prevent his election in 1972 to the presidency of the American Pediatric Society, or to his 1983 Lasker Public Service Award.

To Beecher’s credit, his 1966 paper was instrumental in raising awareness of the need to regulate research using human subjects. Beecher was especially concerned with the protection of children and, apropos that, the nature of informed consent.

In 1974, the National Research Act was signed into law, creating the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. The basic ethical principles identified by the Commission are summarized in its so-called Belmont Report, issued in 1978. Its tenets include minimizing harm to all patients, and the need to especially protect those with “diminished autonomy” or who are incapable of “self-determination.” In addition, federal guidelines now require universities and other research institutions to have Institutional Review Boards to protect human subjects of biomedical research. [Reference 13 (available on line) contains a detailed history of the establishment of these policies.] See Aside 6.

[Aside 6: The infamous U.S. Public Health Service Tuskegee syphilis research program, conducted between 1932 and 1972, in which several hundred impoverished black men were improperly advised and never given appropriate treatment for their syphilis, also raised public awareness of the need to protect human subjects. More recently, research involving embryonic stem cells and fetuses has stoked an ongoing and heated public debate. Policies regarding this research are still not settled, with stem-cell research being legal in some states, and a crime in others. Other recent technological advances, such as DNA identification and shared databases, have been raising new concerns, such as the need to protect patient privacy. In response to these new developments, in June 2016, the US National Academies of Sciences,Engineering and Medicine released a report proposing new rules (indeed a complete overhaul of the 1978 Belmont Report) to deal with these circumstances. The Academy’s report has stirred debate in the biomedical community]

Note 1: The use of children in medical research makes many of us profoundly uneasy. We may be particularly troubled by accounts of the exploitation of institutionalized children, who comprised a uniquely defenseless part of society. Indeed, it was the very vulnerability of those children that made it possible for them to be exploited by researchers. Consequently, some readers may well be asking whether the activities of vaccine researchers Krugman, Koprowski, Sabin, Henle and others might have been comparable to that of the Nazis on trial at Nuremberg. So, I offer this cautionary interjection. While in no way condoning the vaccine researchers using institutionalized children, their work was carried out for the sole purpose of saving human lives. As Koprowski suggested above, if not for that work, we might not have vaccines against smallpox, rabies, yellow fever, and polio. Now, consider Josef Mengele, a Nazi medical officer at Auschwitz, and the most infamous of the Nazi physicians. [Mengele was discussed several times at Nuremberg, but was never actually tried. Allied forces were convinced at the time that he was dead, but he had escaped to South America.] At Auschwitz, Mengele conducted germ warfare “research” in which he would infect one twin with a disease such as typhus, and then transfuse that twin’s blood into the other twin. The first twin would be allowed to die, while the second twin would be killed so that the organs of the two children might then be compared. Mengele reputedly killed fourteen twin children in a single night via a chloroform injection to the heart. Moreover, he unnecessarily amputated limbs and he experimented on pregnant women before sending them to the Auschwitz gas chambers.

References:

Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.

There is a cautionary note on the info sheet accompanying the influenza vaccine, which advises individuals who are allergic to eggs to speak with their doctors before receiving the vaccine. As most readers know, the reason for the warning is that the usual flu vaccine is grown in embryonated chicken eggs.

[Aside 1: The current trivalent influenza vaccine is prepared by inoculating separate batches of fertile chicken eggs; each with one of the three influenza strains (representing an H1N1, an H3N2, and a B strain) recommended by the WHO for the upcoming winter flu season. The monovalent viral yields are then combined to make the trivalent vaccine.]

But, why chicken eggs, and how did this state of affairs come to be? The backdrop to this tale is that until the third decade of the twentieth century, virologists were still searching for fruitful means to cultivate viruses outside of a live laboratory animal. This was so despite the fact that, as early as 1907, researchers had been developing procedures for maintaining viable tissues in culture. And, soon afterwards, virologists began to adapt tissue cultures as substrates for propagating viruses.

Yet as late as 1930, there were still only two antiviral vaccines—the smallpox vaccine developed by Edward Jenner in 1798 (1) and the rabies vaccine developed by Louis Pasteur in 1885. Bearing in mind that Jenner’s vaccine preceded the germ-theory of disease by a half century, and that Pasteur’s vaccine came 15 years before the actual discovery of viruses (as microbial agents that are distinct from bacteria), the development of these first two viral vaccines was fortunate indeed (2).

The principal factor holding up the development of new viral vaccines was that viruses, unlike bacteria, could not be propagated in pure culture. Instead, for reasons not yet understood, viruses could replicate only within a suitable host. And, notwithstanding early attempts to propagate viruses in tissue culture (reviewed below), developments had not yet reached a stage where that approach was fruitful enough to generate a vaccine. How then were Jenner and Pasteur able to produce their vaccines? See Aside 2 for the answers.

[Aside 2: Jenner, without any awareness of the existence of infectious microbes, obtained his initial inoculate by using a lance to pierce a cowpox postule on the wrist of a young milkmaid, Sarah Nelmes. Jenner then propagated the vaccine, while also transmitting immunity, by direct person-to-person transfer. (The rationale underlying Jenner’s vaccine, and his story, is told in detail in reference 1.)

Jenner’s live cowpox vaccine protected against smallpox because cowpox, which produces a relatively benign infection in humans, is immunologically cross-reactive with smallpox. Thus, inoculating humans with cowpox induces immunity that is active against cowpox and against smallpox as well. Jenner’s discovery of the smallpox vaccine, while not entirely fortuitous, was still providential, since immunity per se, as well as microbes, were unknown in Jenner’s day.

Following a successful worldwide vaccination program, smallpox was officially declared to be eradicated in 1977. The smallpox vaccine currently stockpiled in the United States contains live vaccinia; a virus that is immunologically related to cowpox and smallpox. Like cowpox, vaccinia causes a mild infection in humans.

The existing smallpox vaccine was grown in the skin of calves. It is now more than 40 years old and has not been used for years, but it is still believed to be effective.

Pasteur (probably the greatest and most famous microbiologist) was a pioneer of the germ theory of disease. Yet he developed his rabies vaccine more than a decade before the discovery of viruses. He did so by applying the same principle that he used earlier to produce a vaccine against cholera. That is, he “attenuated” the rabies agent. He began with virus that was contained in an extract from a rabid dog. Pasteur attenuated the virus for humans by successively passing extracts in the spinal cords of live rabbits, and then aging the last extracts in the series. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.]

In the years following the pioneering 19th century contributions of Pasteur, Koch, and Lister, and with the widespread acceptance of the germ theory of disease, microbiologists (that is, bacteriologists) appreciated the importance of working with “pure cultures” that could be grown in a sterilized medium. Yet this was proving to be impossible in the case of viruses. Moreover, as late as the 1930s, it was not understood why that should be so

At the very least, virologists would have liked to be able to cultivate viruses outside of a living animal host. The possibility of achieving that goal began to emerge when Ross G. Harrison, working at Johns Hopkins in 1907, became the first researcher to maintain bits of viable tissue outside of an animal. Harrison maintained frog neuroblasts in hanging drops of lymph medium. What’s more, under those conditions, the neuroblasts gave rise to outgrowths of nerve fibers.

In 1913, Edna Steinhardt became the first researcher to cultivate (or at least maintain) a virus (cowpox) in a tissue culture. Steinhardt did this by infecting hanging-drop cultures with corneal extracts from the eyes of cowpox-infected rabbits and guinea pigs. However, there was no methodology at the time for Steinhardt to determine whether the virus might have replicated in her tissue cultures.

In 1912, Alexis Carrel, working at the Rockefeller Institute, began a two-decade-long experiment that significantly increased interest in tissue culture. Carrel maintained tissue fragments from an embryonic chicken heart in a closed flask, which he regularly supplied with fresh nutrients. Later, he claimed that he maintained the viability of the culture for more than 20 years; well beyond the normal lifespan of a chicken. See Aside 3.

[Aside 3: Carrel’s experimental results could never be reproduced. In fact, in the 1960s, Leonard Hayflick and Paul Moorhead made the important discovery that differentiated cells can undergo only a limited number of divisions in culture before undergoing senescence and dying. It is not known how Carrel obtained his anomalous results. But, Carrel was an honored, if controversial scientist, having been awarded the 1912 Nobel Prize in Physiology or Medicine for pioneering vascular suturing techniques. In the 1930s Carrel developed an intriguing and close friendship with Charles Lindbergh, which began when Lindbergh sought out Carrel to see if Carrel might help Lindbergh’s sister, whose heart was damaged by rheumatic fever. Carrel could not help Lindbergh’s sister, but Lindbergh helped Carrel build the first perfusion pump, which laid the groundwork for open heart surgery and organ transplants. Carrel and Lindbergh also co-authored a book, The Culture of Organs. In the 1930s, Carrel, promoted enforced eugenics. During the Second World War, Carrel, who was French by birth, helped the Vichy French government put eugenics policies into practice. Moreover, he praised the eugenics policies of the Third Reich, leading to inconclusive investigations into whether he collaborated with the Nazis. Carrel died in November, 1944.]

In 1925 Frederic Parker and Robert Nye, at the Boston City Hospital, provided the first conclusive evidence for viral growth in a tissue culture. The virus was a strain of herpes simplex, which Parker and Nye received in the form of an extract from Ernest Goodpasture; soon to be the major character in our story. Parker and Nye established their first culture from the brain of a rabbit that was inoculated intracerebrally with an extract from an infected rabbit brain. The animal was sacrificed when in a convulsive state, and its brain was then removed aseptically. Small pieces of normal rabbit testes were added to pieces of brain in the cultures, to provide another potential host cell for the virus. Virus multiplication was demonstrated by inoculating diluents of subculture extracts into laboratory animals. A 1:50,000 diluent was able to transmit the infection.

At this point in our chronology, the pathologist Ernest Goodpasture, and the husband-wife team of Alice and Eugene Woodruff, enters our story. Goodpasture’s principal interest was then, as always, in pathology. He became interested in viruses while he was serving as a Navy doctor during World War I. But his focus was on the pathology of the 1918 influenza pandemic, which he studied in the first sailors stricken by the infection (3). He was later interested in herpetic encephalitis, and in how rabies virus made its way to the central nervous system, but always from the perspective of a pathologist.

Ernest Goodpasture. (I was unable to find a picture of Alice Woodruff.)

In 1927, Eugene Woodruff was a newly graduated physician who joined Goodpasture in the Pathology Department at Vanderbilt University for training as a pathologist. Eugene’s wife, Alice, a Ph.D., came to the Vanderbilt Pathology Department a year later, as a research fellow in Goodpasture’s laboratory.

Goodpasture set Eugene Woodruff to work on fowlpox; a relative of smallpox, which, unlike cowpox, can not infect humans. Goodpasture was interested in the cellular pathology of fowlpox infection; specifically, in the nature of the inclusion bodies seen in fowlpox-infected cells. Using a micropipette, Woodruff was able to pick single inclusion bodies from infected chicken cells, and to then determine that inclusion bodies are intracellular crystalline arrays of the virus.

More apropos to our story, in the late 1920s, virologists still could not generate large amounts of virus that were free of bacteria and contaminating tissue elements. For that reason, Goodpasture believed that future important advancements in virology would require the development of methods to grow large amounts of virus in pure culture; an impossible goal. In any case, Goodpasture delegated Alice Woodruff to develop a method for growing fowlpox outside of a live chicken.

Goodpasture had already adapted Carrel’s tissue culture methods, which he used to maintain chick kidney tissue in culture. So, Alice’s first experiments were attempts to get fowlpox to propagate in cultures of chick kidney tissue. However, the virus stubbornly declined to grow in the tissue cultures. Goodpasture then suggested to Alice that she try to grow the virus in embryonated chicken eggs. But why did Goodpasture make that suggestion?

The answer isn’t clear. But, back in 1910, Peyton Rous and colleague James Murphy, at the Rockefeller Institute, fruitfully made use of fertile chick eggs to cultivate a virus, as described in Aside 4. However, Rous’ accomplishments, which eventually would be recognized as huge, were largely ignored for the next 50 or so years. (The reasons are discussed in reference 4.) Goodpasture may well have been unaware of Rous’ earlier work when he suggested to Alice that she try to cultivate fowlpox in chicken eggs. If so, then his suggestion to Alice may have been an original idea on his part, perhaps inspired by his thinking of the chick embryo as a sterile substrate that is enclosed in a naturally sterile container. On the other hand, he and Alice did note the earlier work of Rous and Murphy in the 1931 report of their own work. (In that paper, they state: “The production of experimental infection in the chorio-allantoic membrane has, however, been done only in the one instance where Rous and Murphy grew the virus of the Rous sarcoma.”). In any case, the chick embryo method for growing viruses had lain dormant for twenty years.

[Aside 4: Rous and Murphy cut a small window into the shells of six-to-sixteen-day-old embryonated chicken eggs, and then placed a bit of a filtered, cell-free extract from a chicken sarcoma into each. By one week’s time there was a tumor mass growing in each of the inoculated embryos. These studies led to Rous’ 1911 report of a filterable, infectious agent, eventually named the Rous sarcoma virus, which causes sarcomas in chickens. The Rous sarcoma virus was the first virus known to cause solid tumors and, moreover, it was the prototype of a virus family that eventually would be known as the retroviruses (4).]

Alice Woodruff’s procedure for infecting the chicken eggs began with her making a small window in the egg shell, at the site of the air sac. (An egg cup served as the operating table, and the window was cut with a dentist’s drill.) She then inoculated the viral extract into the outermost layer of the chorio-allantoic membrane, which encloses the embryo and provides an air channel into its body. Alice then closed the window with a piece of glass, held in place with Vaseline.

Alice tried to maintain sterility at all stages of her procedure. Yet despite the elegance of her techniques, she had nothing to show for these efforts except dead embryos that were overgrown with mold or bacteria. She then turned to her husband, Eugene, who was working in a separate laboratory, down the hall from her lab.

Alice and Eugene, working together, developed procedures to sterilely remove fowlpox lesions from the heads of chicks. In brief, the chick heads were shaved and then bathed in alcohol. Then, the lesions were excised with sterile instruments. Next, the excised lesions were tested for bacterial or fungal contamination by incubating fragments in nutrient broth. If a lesion was sterile by that test, it was deemed fit to be inoculated into the eggs.

Eugene further contributed to the effort by applying a technique that he developed earlier; picking out individual inclusion bodies from fowlpox-infected cells. When he discovered that the inclusion bodies could be disrupted into individual virus particles by incubating them in trypsin, he was able to provide Alice with virtually pure virus that she could inoculate the eggs with.

As Greer Williams relates in Virus Hunters (5): “Then, one morning when she peeked into the window of an egg that had been incubating for about a week after she had infected it with the virus, she saw something different. This chick embryo was still alive…She removed the embryo from the shell and examined it. It had a swollen claw. ‘Could this be due to fowlpox infection?’…She went to Goodpasture and put the same question to him…”

In Alice’s own words, “I can’t forget the thrill of that moment when Dr. Goodpasture came into my lab, and we stood by the hood where the incubator was installed and I showed him this swollen claw from the inoculated embryo (5).”

The swollen claw indeed resulted from the fowlpox infection. This was shown by the fact that when bits of the swollen tissue were transferred to other embryos, they in turn induced more swollen tissue. Moreover, these swollen tissues contained fowlpox inclusion bodies. Additionally, when transferred to adult chickens, those bits of swollen tissue produced typical fowlpox lesions.

During the next year, Goodpasture, Alice Woodruff, and Gerritt Budding (a lab assistant, who dropped out of medical school to participate in the chick embryo work) reported that cowpox and herpes simplex viruses could also be grown in the embryonated chicken eggs.

Later studies by Goodpasture and Buddingh showed that each embryonated chicken egg could produce enough vaccinia to produce more than 1,000 doses of smallpox vaccine. They also showed, in a case-study involving 1,074 individuals, that the chick-grown smallpox vaccine works as well in humans as the vaccine produced by inoculating the skin of calves. Regardless, the chick vaccine never caught on to replace the long-established, but cruder calf-grown vaccine (see Aside 2).

Goodpasture placed Alice’s name ahead of his own on their report describing the propagation of fowlpox in chicken eggs. Alice says that Goodpasture was “over-generous” in that regard. Howevever, much of the day-to-day lab work resulted from her initiatives. Eugene’s name also came before Goodpasture’s on the report describing the inclusion body study.

Shortly after completing these studies, Alice left research to raise a family. Eugene’s name also disappeared from the virus literature. But in his case that was because his interests turned to tuberculosis.

In 1932, soon after the above breakthroughs in Godpasture’s laboratory, Max Theiler and Eugen Haagen developed their yellow fever vaccine (6), which initially was generated in embryo tissue from mice and chickens. But, starting in 1937, production of the yellow fever vaccine was switched to the embryonated egg method, in part, to “cure” the live yellow fever vaccine of its neurotropic tendencies.

Recall our introductory comments regarding the warning that individuals allergic to eggs should get medical advice before receiving the standard flu vaccine. In 1941, Thomas Francis, at the University of Michigan, used embryonated chicken eggs to produce the first influenza vaccine (see Asides 5 and 6). Remarkably, even today, in the era of recombinant DNA and proteomics, this seemingly quaint procedure is still the preferred means for producing the standard trivalent flu vaccine (see Aside 1).

[Aside 5: Thomas Francis produced his 1941influenza vaccine in response to urging by U.S. Armed Forces Epidemiological Board. With the Second World War underway in Europe and Asia, and with the 1918 influenza pandemic in mind, there was fear that if an influenza epidemic were to emerge during the upcoming winter, it might impede the military training that might be necessary. An epidemic did not materialize that winter, but the vaccine was ready, and we were at war.]

[Aside 6: Thomas Francis was one of the great pioneers of medical virology. The same year that he developed his flu vaccine, Jonas Salk (recently graduated from NYU medical school) came to his laboratory for postgraduate studies. Francis taught Salk his methodology for vaccine development, which ultimately enabled Salk to develop his polio vaccine (7).]

Next, Hillary Koprowski developed a safer, less painful and more effective rabies vaccine that is grown in duck eggs, and that is still widely used. Why duck eggs? The reason is that duck eggs require four weeks to hatch, instead of the three weeks required by chicken eggs. So, duck eggs give the slow-growing rabies virus more time to replicate.

By any measure, the procedures for growing viruses in embryonated chicken eggs, developed by Ernest Goodpasture and Alice Woodruff, were a major step forward in vaccine development. Sir Macfarlane Burnet (a Nobel laureate for his work on immunological tolerance) commented 25 years later, “Nearly all the later practical advances in the control of viral diseases of man and animals sprang from this single discovery.”

Addendum 1: Several major advances in cell and tissue culture (the other means for growing viruses outside of an animal) happened after Woodruff and Goopasture reported the development of their embryonated egg method in 1931. For the sake of completeness, several of these are noted.

In 1933, George Gey, at Johns Hopkins, developed the roller tube technique, in which the tissue is placed in a bottle that is laid on its side and continuously rotated around its cylindrical axis. In that way, the media continually circulates around the tissue. Compared to the older process of growing tissues in suspension, the roller culture method allowed the prolonged maintenance of the tissues in an active state and, consequently, the growth of large amounts of virus. The roller tube technique also works very well for cell cultures that attach to the sides of the bottle. [Incidentally, Gey is probably best known for having established the HeLa line of human carcinoma cells from cancer patient, Henrietta Lacks. HeLa cells comprise the first known human immortal cell line and they have served as one of the most important tools for medical research. (See The Immortal Life of Henrietta Lacks, by Rebecca Skloot, 2010.)]

In 1948, John Enders, and colleagues Thomas Weller and Frederick Robbins, used Gey’s methods, to demonstrate for the first time that poliovirus could be grown in non-nervous tissue. This was significant because the potential hazard of injecting humans with nervous tissue was holding up the development of a polio vaccine.

Next, Renato Dulbecco and Marguerite Vogt, working at Caltech, developed procedures to grow large amounts poliovirus in cell culture, adding to the feasibility of an eventual polio vaccine (8). Additionally, Dulbecco and Vogt developed a plaque assay procedure to measure the titer of animal viruses grown in cell culture (7).

Addendum 2: The following excerpt tells of the chance encounter that led Howard Temin to become a virologist (4). Temin was the Nobel laureate who first proposed the retroviral strategy of replication, and who co-discovered reverse transcriptase.

“Howard Temin began working on Rous sarcoma virus in the 1950s, while a graduate student in Renato Dulbecco’s laboratory at Caltech (see reference 7 for more on Dulbecco). However, he worked under the direct supervision of Harry Rubin, an early star in the field, who was, at the time, a postdoctoral fellow in the Dulbecco lab. Nothing was known as yet about the replication of the RNA tumor viruses, as the retroviruses were then known. Moreover, little more was known about the molecular basis of cancer in the 1950s than was known in 1911, when Rous first isolated his virus; a state of affairs that would be much alleviated by future studies of the oncogenic retroviruses.

Rubin was a veterinarian by training, perhaps accounting for his somewhat unique appreciation of an oncogenic virus of chickens, well after even Rous himself had lost interest. And, Rubin was responsible for introducing other young investigators to the RNA tumor virus field, both at Caltech and later at UC Berkely.

Rubin’s mentorship of Temin began somewhat fortuitously, as follows. When they first met, Temin was actually doing his graduate research in another laboratory at Caltech, looking into the embryology of the innkeeper worm, Urechis caupo. But he was also serving as a laboratory assistant in the Caltech general biology course. In that capacity, he was dispatched to Dulbecco’s laboratory to obtain some fertilized chicken eggs for use in the general biology lab. Harry Rubin supplied the chicken eggs. But the chance visit from Temin gave Rubin the opportunity to tell Temin about the chicken sarcoma viruses that were being studied in the Dulbecco laboratory.

Rubin had just recently found that he could induce the neoplastic transformation of a normal chicken cell with a single Rous sarcoma virus particle. He then demonstrated that the transformed cell produced hundreds more transformed daughter cells in a week’s time. During their chance conversation, Rubin suggested to Temin that he (Temin) might make use of that observation to develop a quantitative tissue culture assay for Rous sarcoma virus. Sufficiently intrigued by Rubin’s proposition, Temin switched from embryology to virology and proceeded to develop a focus-forming cell culture assay for Rous sarcoma virus; an assay analogous in principle to a plaque assay. But instead of forming plaques of dead cells, the non-cytocidal Rous sarcoma virus induces the growth of visible foci of morphologically transformed neoplastic cells.”

[Addendum 3: Today, viruses are usually cultivated in readily available continuous cell lines. That said, when I first entered the field in 1970, as a postdoctoral studying the murine polyomavirus, my first task of the week was to prepare the baby-mouse-kidney and mouse-embryo primary cell cultures, which at that time served as the cellular host for that virus. This rather unpleasant chore was a reason I eventually turned to SV40, since I could grow that virus in continuous lines of monkey kidney cells.

References:

1. Edward Jenner and the Smallpox Vaccine, posted on the blog September 16, 2014.

The first part of this posting tells how a U.S. Army medical board, headed by Walter Reed, confirmed that the transmission of yellow fever requires a mosquito vector. The second part tells the story of the yellow fever vaccine developed by Max Theiler.

Bearing in mind the enormous benefit to mankind of the polio vaccines developed by Jonas Salk and Albert Sabin (1), and that Maurice Hilleman developed nearly 40 vaccines, including those for measles, mumps, and rubella (2), it would appear remarkable that Theiler was the only one of these four individuals to be recognized by the Nobel committee. In fact, Theiler’s 1951 Nobel award was the only one ever given for a vaccine! In any case, while Theiler’s vaccine was a major step forward in the fight against yellow fever, it came after a perhaps more dramatic episode in the struggle against that malady. But first, we begin with some background.

Yellow fever was another of mankind’s great scourges. Indeed, it was once the most feared infectious disease in the United States. And, while we might want to say that science has “conquered” yellow fever, that statement would not be entirely accurate. Unlike polio and measles, which have nearly been eradicated by the vaccines against them, that is not so for yellow fever. The reason is as follows. Humans are the only host for polio and measles viruses. Consequently, those viruses might be completely eradicated if a sufficient percentage of humans were to comply with vaccination regimens. In contrast, the yellow fever virus infects monkeys that range over thousands of square miles in Africa and the Amazon jungle. Thus, even with massive vaccination of humans, it would be impossible to eliminate the yellow fever virus from the world.

According to the World Health Organization’s estimates, there are still about 200,000 cases of yellow fever per year, resulting in about 30,000 deaths, about 90% of which occur in Africa. The yellow fever virus itself is the prototype virus of the flavivirus family of single-stranded RNA viruses, which also includes dengue hemorrhagic fever virus, Japanese encephalitis virus, and West Nile encephalitis virus, among others.

Yellow fever is somewhat unique among the viral hemorrhagic fevers in that the liver is the major target organ. Consequently, the severe form of yellow fever infection is characterized by hemorrhage of the liver and severe jaundice. But, as in infections caused by other virulent viruses, most cases of yellow fever are mild.

Interestingly, the name “yellow fever” does not have its origin in the yellowing of the skin and eyes that is characteristic of severe disease. Instead, it has its origin in the term “yellow jack,” which refers to the yellow flag that was flown in port to warn approaching ships of the presence of infectious disease.

Yellow fever originated in Africa. It is believed to have been brought to the New World by slave ships in the year 1596. As noted above (and discussed below), yellow fever transmission, from an infected individual or primate to an uninfected one, requires a specific vector, the Aedes aegypti mosquito. The sailing ships of the day inadvertently transported the disease across oceans via the mosquito larvae in their water casks.

Before getting to our stories proper, we note a pair of intriguing instances in which yellow fever profoundly affected New World history. In the first of these, yellow fever was a key factor that led Napoleon to sell the Louisiana Territory to the United States in 1803; an act that doubled the size of the United States. It happened as follows. After Napoleon seized power in France, he reinstated slavery in the French colony of Saint Domingue (now Haiti); doing so for the benefit of the French plantation owners there. In response, the rather remarkable Toussaint Breda (later called Toussaint L’Ouverture, and sometimes the “black Napoleon”) led a slave revolt against the plantation owners. In turn, in February 1802, Napoleon dispatched an expeditionary force of about 65,000 men to Haiti to put down the revolt. The rebellious slaves, many fewer in number than the French, cleverly retreated to the hills, believing that the upcoming yellow fever season would wreak havoc on the French force. And, they were correct. By November 1803, the French lost 50,000 of the original 65,000 men to yellow fever and malaria. Thus, in 1804, Napoleon had to allow Haiti to proclaim its independence, and then become the second republic in the Western Hemisphere. Moreover, there is evidence suggesting that Napoleon’s actual purpose in dispatching the expeditionary force was to secure control of France’s North American holdings. With his expeditionary force decimated by yellow fever and malaria, that was no longer possible and, consequently, Napoleon sold France’s North American holdings (the Louisiana Purchase) to the United States.

The Louisiana Purchase, in green.

Second, in 1882, France began its attempt to build a canal across the Isthmus of Panama. However, thousands of French workers succumbed to yellow fever, causing France to abandon the project. The United States was able to successfully take up the task in 1904; thanks to the deeds of the individuals in part I of our story, which now begins.

In May 1900, neither the cause of yellow fever, nor its mode of transmission was known. At that time, U.S. Army surgeon, Major Walter Reed, was appointed president of a U.S. Army medical board assigned to study infectious diseases in Cuba, with particular emphasis on yellow fever. Cuba was then thought to be a major source of yellow fever epidemics in the United States; a belief that was said to have been a factor in the American annexation of Cuba.

Major Walter Reed

When Reed’s board began its inquiry, a prevailing hypothesis was that yellow fever might be caused by the bacterium Bacillus icteroides. However the board was unable to find any evidence in support of that notion.

Another hypothesis, which was advanced by Cuban physician Dr. Carlos Juan Finlay, suggested that whatever the infectious yellow fever agent might be, transmission to humans requires a vector; specifically, the mosquito now known as Aedes aegypti. Reed was sympathetic to this idea because he noticed that people who ministered to yellow fever patients had no increased risk of contracting the disease, which indicated to Reed that people did not pass yellow fever directly from one to another.

Reed, as president of the medical board, is generally given major credit for unraveling the epidemiology of yellow fever. Yet there were other heroes in this story as well. Finlay, whose advice and experience were invaluable to Reed’s board, was one. He was the object of much ridicule for championing the mosquito hypothesis, at a time when there little evidence that might support it. In any case, Reed, in his journal articles and personal correspondences, gave full credit to Finlay for the mosquito hypothesis.

Acting Assistant Surgeon Major James Carroll was another hero. As a member of Reed’s board, Carroll volunteered to be bitten and, promptly, developed yellow fever. Major Jesse Lazear, also a board member, asked Private William Dean if he might be willing to be bitten. Dean consented, and he too contracted yellow fever. Fortunately, Dean and Carroll each recovered. Not so for Lazear. After allowing himself to be bitten, he died after several days of delirium.

Lazear’s contribution to gaining recognition of the mosquito hypothesis went significantly beyond his tragic martyrdom. When Reed examined Lazear’s notebook after his death, Reed found that it contained several key observations. First, Lazear had carefully documented that in order for a mosquito to be infected; it had to bite a yellow fever patient within the first three days of the patient’s illness. Second, twelve days then had to elapse before the virus could reach high enough levels in the insect’s salivary glands to be transmitted to a new victim.

The observations of the board, up to then, convinced Reed and the others that the mosquito hypothesis indeed was correct. Yet Reed knew that more extensive controlled experiments would be needed to convince the medical community. So, he directly supervised those experiments, which involved twenty-four more volunteers, each of whom may rightly be considered a hero.

Just as Reed benefited from Finlay’s insights, William C. Gorgas, Surgeon General of the U.S. Army, applied the findings of Reed’s board to develop vector control measures to combat urban yellow fever; first in Florida, then in Havana, Cuba, and next in Panama, where those measures enabled the United States to complete the canal in 1914. The last urban yellow fever outbreak in the United States occurred in New Orleans in 1905, and the last in the New World occurred in 1999 in Bolivia.

The vector control strategy works for urban yellow fever because the Aedes aegypti mosquitoes have a very short flight range and, consequently, the female mosquito does not stray far from the source of her blood meal before laying her eggs. Thus, it is only necessary to control the vector population in the immediate vicinity of human habitation. In practice, this is accomplished by draining potential mosquito breeding sites such as swamps and ditches, and destroying water-collecting objects such as discarded tires.

After Reed’s board was disbanded, he made yet another key contribution to the wiping out of yellow fever. The focus of the board had been on the means of yellow fever transmission; not with the infectious agent itself. In 1901, at the suggestion of William Welch, an eminent Johns Hopkins pathologist, Reed and James Carroll (who nearly died of yellow fever after being experimentally infected while in Cuba), asked whether yellow fever might be caused by a filterable virus. Indeed, they found that they could infect volunteers by inoculating them with filtered serum taken from yellow fever patients. What’s more, theirs was the very first demonstration of a human illness being caused by a filterable agent. That is, yellow fever was the first human illness shown to be caused by a virus. [Pasteur developed an attenuated rabies vaccine in 1885, more than a decade before the discovery of viruses. Remarkably, this most brilliant of experimentalists did not recognize that he was dealing with a previously unknown, fundamentally distinct type of infectious agent; the topic of a future posting.]

[Aside: Walter Reed spent the early years of his Army career at different posts in the American west. The Mount Vernon Barracks in Alabama, which served as a prison for captured Apache Native Americans, including Geronimo, was a particularly interesting stop for Reed. Captain Walter Reed, serving as post surgeon in the 1880s, looked after Geronimo and his followers.]

Part II of this posting concerns the development of Max Theiler’s yellow fever vaccine. But first, here is a bit more background.

Vector control measures ended yellow fever epidemics in most, but not all urban centers worldwide. Outbreaks have not occurred in the United States for more than a century. However, jungle yellow fever still persists in areas of Sub-Saharan Africa and, to a lesser extent, in tropical South America. Individuals who are infected in the jungle by wild mosquitoes can then carry the virus to densely populated urban areas, where Aedes aegypti mosquitoes can transmit the virus from one individual to another. [Vector-mediated, human-to-human transmission happens because the level of yellow fever virus in the blood of an infected person becomes high enough for the infected person to transmit the virus to a biting mosquito. In this regard, the yellow fever virus is an exception to the generalization that humans are a “dead end” host for arthropod-borne (arbo) viruses.]

Fortunately, people who live in high risk areas for yellow fever can be protected by vaccination. Indeed, the World Health Organization’s strategy for preventing yellow fever epidemics in high risk areas is, first, to mass immunize the population, and then to routinely immunize infants. [Vaccinated American or European visitors to West Africa or the Amazon need not be concerned about yellow fever. However, the risk to an unvaccinated person of acquiring yellow fever during a two-week stay at the height of the transmission season (July through October), is estimated to be 5%. Individuals wanting to enter or return from countries where yellow fever is endemic may need to show a valid certificate of vaccination. ]

Part II of our story, concerning Max Theiler and the development of the yellow fever vaccine now begins.

Even as late as the 1920s, some reputable bacteriologists remained unconvinced by the earlier findings of Reed and Carroll that yellow fever is caused by a filterable agent. Instead, they persisted in the belief that the illness is caused by a bacterium. The notion of a bacterial etiology for yellow fever was finally put to rest after A. H. Mahaffy in 1927 discovered that the yellow fever agent could be propagated and cause illness in Asian rhesus monkeys. With an experimental animal now at hand, yellow fever workers were able to prove conclusively that the disease is caused by a virus. [Mahaffy drew the virus he used in his experiments from a 28-year-old African man named Asibi, who was mildly sick with yellow fever. That isolate, referred to as the Asibi strain, will play an important role later in this anecdote.]

Regardless of the significance of the discovery that the yellow fever virus could be propagated in rhesus monkeys, Max Theiler had to contend with the fact that these monkeys were quite expensive; especially for a not yet established young investigator. [They cost the then princely sum of about $7.00 apiece.] As for mice, while they could be bred for pennies apiece, other researchers were not able infect them via the usual practice of inoculating them under the skin or in the abdomen. However, Theiler took a cue from Pasteur’s inability to propagate the rabies virus in laboratory rabbits until he put the virus directly into their brains. Thus, in 1929 Theiler attempted to do the same with yellow fever virus in mice.

Max Theiler

Theiler’s attempts to infect the mice by intracranial injection were a success. All of the inoculated mice died within several days. Surprisingly, the dead mice did not display the liver or renal pathology characteristic of yellow fever. Instead, the mice appeared to have succumbed to inflammation of their brains. Thus, the yellow fever virus appeared to be neurotropic in mice. Also, Theiler himself contracted yellow fever from one of his inoculated mice. He was fortunate to survive.

A fortuitous result of Theiler’s perilous bout with yellow fever was that he had become immune to the virus, as revealed by the presence of antiviral antibodies in his blood. Importantly, Theiler’s acquired immunity to the virus validated the possibility of developing an attenuated yellow fever vaccine. And, in a sense, Theiler was inadvertently the first recipient of the nascent vaccine he soon would be developing.

Theiler also determined that the virus could be passed from one mouse to another. And, while the virus continued to cause encephalitis in mice, it caused yellow fever when inoculated back into monkeys; quite a unique and striking set of findings. But, and crucially significant, while continued passage of the virus in mice led to its increased virulence in those animals, the virus was concurrently losing its virulence in monkeys. [In 1930, Theiler moved from the Harvard University School of Tropical Medicine to the Rockefeller Foundation’s Division of Biological and Medical Research. The Rockefeller Foundation shared facilities with the Rockefeller Institute (now University); although it was otherwise administratively separate from it.]

Since the mouse-passed virus was becoming attenuated in monkeys, Theiler’s belief in the possibility of generating an attenuated yellow fever vaccine was bearing out. However, because the mouse-passed virus remained neurovirulent in mice, Theiler was reluctant to inoculate that virus into humans. In an attempt to solve this problem, Theiler turned from passing the virus in the brains of live mice and, instead, began passing the virus in mouse tissue cultures.

Theiler carried out seventeen different sets of trials to further attenuate the virus. In the 17th of these, Theiler used the wild Asibi strain, isolated earlier by Mahaffy. Initially, this virus was extremely virulent in monkeys, in which it caused severe liver damage. But, after passing the virus from culture to culture several hundred times, over a period of three years, a flask labeled 17D yielded the virus that was to become the famous 17D yellow fever vaccine.

Theiler never gave a satisfactory accounting for the “D” in the “17D” designation, and for what, if anything became of A, B, and C. Regardless, the genesis of 17D was as follows. Theiler initially took an Asibi sample that had been multiplying in mouse embryo tissue and continued passing it in three separate types of minced chicken embryo cultures. One of these sets contained whole minced chicken embryos, and was designated 17D (WC). A second set contained chick embryo brain only, and was designated 17D (CEB). In the third set, the brains and spinal cords were removed from the otherwise whole chick embryo tissue cultures. This set, alone among all the sets, generated an attenuated virus that did not induce encephalitis when injected directly into monkey brains. Indeed, Theiler removed the central nervous systems from the chicken tissue in this set of cultures, in the express hope of generating just such an attenuated virus. And, by hook or by crook, the virus emerging from that particular set of passages became the vaccine that is now known simply as 17D.

Field tests of Theiler’s yellow fever vaccine were underway in 1937 in Brazil, and were successfully completed by 1940. In 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for developing the vaccine.

Next, we return to a point noted above, and discussed in two earlier postings. Neither Jonas Salk nor Albert Sabin were awarded Nobel prizes for developing their polio vaccines (1). And, Maurice Hilleman was never awarded a Nobel Prize, despite having developed nearly 40 vaccines, including those for measles, mumps, and rubella (2). Indeed, Max Theiler’s Nobel Prize for the yellow fever vaccine was the only Nobel Prize ever awarded for a vaccine! Why was that so?

Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. [Hilleman’s basic discoveries regarding interferon should have been sufficient to earn him the award (2). The slight to him may have been because the Nobel committee was reluctant to give the award to an “industrial” scientist. Hilleman spent the major part of his career at Merck & Co.]

So, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur similarly developed an attenuated Rabies vaccine in 1885?

Perhaps the “discovery” was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryo cultures, which were devoid of nervous system tissue, generated attenuated yellow fever virus that was no longer neurovirulent in mice and monkeys. But, consider the following.

Theiler indeed believed that removing the brains and spinal cords from the chick embryo cultures in which 17D had been serially passed was the reason why the virus lost its neurovirulence. Nevertheless, as a serious scientist he needed to confirm this for himself. So, he repeated the long series of viral passages under the same conditions as before. But, this time, there was no loss of neurovirulence. Thus, a cause and effect relationship, between the absence of the brains and spinal cords from the tissue cultures and the emergence of non-neurovirulent virus, was not confirmed.

So, perhaps the Nobel committee merely paid lip service to the directives in Alfred Nobel’s will. In any case, Theiler’s 17D yellow fever vaccine has had a virtually unblemished safety record, and is regarded as one of the safest and most effective live-attenuated viral vaccines ever developed.

Theiler’s unshared 1951 Nobel award paid him $32,000. At the time, he resided in Hastings-on-Hudson; a village in Westchester County, NY, from which he commuted to the Rockefeller labs. Theiler’s next door neighbor in Hastings-on-Hudson was Alvin Dark, the star shortstop of the New York Giants. Nobel laureate Max Theiler was known to fellow commuters from Hastings-on-Hudson as the man who lives next door to Alvin Dark.

Virus Hunters, by Greer Williams (Alfred A, Knoff, 1960) was my major source for the material on Max Theiler.

1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science. On the blog.

In January 2005, more than 100 of the world’s most renowned biomedical researchers got together to pay tribute to the 85-year-old Maurice Hilleman. When it was Hilleman’s turn to address the gathering, he alluded to them as his “peers in the world of science.” Referring to Hilleman’s gracious comment, science journalist Alan Dove wrote: “By any objective measure, a gathering of Maurice Hilleman’s scientific peers would not fill a telephone booth.” (1)

Hilleman truly was a giant in the history of virology. But, if you have only a vague idea of who Hilleman was or of his achievements, you are not alone. Anthony Fauci, director of the U.S. National Institutes of Allergy and Infectious Diseases, who was present at the gathering, noted: “Very few people, even in the scientific community, are even remotely aware of the scope of what Maurice has contributed….I recently asked my post-docs whether they knew who had developed the measles, mumps, rubella, hepatitis B and chickenpox vaccines. They had no idea,” Fauci said. “When I told them that it was Maurice Hilleman, they said, ‘Oh, you mean that grumpy guy who comes to all of the AIDS meetings?’”

Maurice R. Hilleman: The greatest vaccinologist.

Consider this. Hilleman developed nine of the 14 vaccines routinely recommended in current vaccine schedules. These are the vaccines for the measles, mumps, rubella, hepatitis A, hepatitis B, and chickenpox viruses, and for meningococcal , pneumococcal, and Haemophilus influenzae bacteria. Moreover, he was the first to forecast the arrival of the 1957 Asian flu and, in response, led the development of a flu vaccine that may have saved hundreds of thousands or more lives worldwide (2). And, independently of Robert Huebner and Wallace Rowe, he discovered cold-producing adenoviruses, and developed an adenovirus vaccine. Overall, Hilleman invented nearly 40 vaccines. And, he was a discoverer of simian virus 40 (SV40). If the above accomplishments were not enough to ensure his fame, he also was the first researcher to purify interferon, and the first to demonstrate that its expression is induced by double-stranded RNA.

[Aside: I first became aware of Maurice Hilleman 44 years ago. It was in the context of his 1959 discovery of SV40, which I came across only because I was beginning my post-doctoral studies of the related murine polyomavirus. Bernice Eddy, at the U. S. National Institutes of Health (NIH), was probably the first to discover SV40, which she detected in early lots of the Salk polio vaccine (3). Hillman, then at Merck & Co, independently discovered the same virus in rhesus monkey kidney cell cultures, in which the polio vaccine was being produced. Hilleman gave SV40 its name. It was the 40th simian virus the Merck lab found in the monkey kidney cells. In 1961, both Eddy and Hilleman found that inoculating SV40 into hamsters causes tumors in the animals. Merck withdrew its polio vaccine from the market. But, by then, live SV40 had been unknowingly injected into hundreds of millions of people worldwide! More on this in a future posting.]

We begin our account of Hilleman’s achievements with his development of the mumps vaccine. In the days before the vaccine, mumps struck about 200,000 children in the United States, annually. Yet except in rare circumstances, the infection was mild, and was generally regarded as a childhood rite of passage. There is a sweetness to the story of the mumps vaccine that I hope you might enjoy.

The tale began at about 1:00 AM, on March 21, 1963, when 5-year-old Jeryl Lynn Hilleman ambled into her father’s bedroom complaining of a sore throat. Jeryl Lynn’s father felt his daughter’s swollen glands, and knew in a flash that it was mumps. And, while I suspect that many lay parents back in the day would also have recognized Jeryl Lynn’s symptoms, few would have done what her father did after first comforting his daughter. Although it was already past midnight, Maurice hopped into his car and drove the 20 minutes to his lab at Merck & Co. to pick up some cotton swabs and beef broth. Returning home, he then awakened Jeryl Lynn, gently swabbed her throat, and immersed the swabs in the nutrient broth. Next, he drove back to his lab and put the inoculated broth in a freezer.

Hilleman made the early A.M. dashes to his lab and back because he had to leave in the morning for a conference in South America, and his daughter’s infection might have cleared by the time he returned home from there. So, upon his return from South America, Hilleman, thawed the frozen sample from his daughter’s throat and inoculated it into chick embryos. Serial passage of the mumps virus in the chick embryos eventually generated attenuated mumps virus that in 1967 would serve as a live mumps vaccine.

The virus in the vaccine was dubbed the Jeryl Lynn strain, in honor of its source. Years later, an adult Jeryl Lynn Hilleman noted that her father had a need to be “of use to people, of use to humanity.” She added: “All I did was get sick at the right time, with the right virus, with the right father.”

We’ll have a bit more to say about the mumps vaccine shortly. But first, a few words about measles and rubella.

If mumps was not a major killer, measles certainly was. Before Hilleman and his colleagues introduced their measles vaccine (Rubeovax) in 1962, there were 7 to 8 million measles fatalities worldwide each year, and virtually all of the victims were children. Hilleman developed his attenuated measles vaccine from a measles strain isolated earlier by John Enders. Hilleman attenuated the Enders isolate by putting it through 80 serial passages in different cell types.

[Aside: In a previous posting, we noted that Enders, together with colleagues Thomas Weller and Frederick Robbins, shared a Nobel Prize in Physiology or Medicine for growing poliovirus in non-nervous tissue (3). Apropos the current story, bear in mind that Salk and Sabin developed polio vaccines that have nearly rid the world of this once dread virus. Nevertheless, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research!]

Rubeovax was somewhat tainted by its side effects; mainly fever and rash. While these reactions were successfully dealt with by combining Rubeovax with a dose of gamma globulin, in 1968 Hilleman’s group developed a new, more attenuated measles strain by passage of the Rubeovax virus 40 more times through animal tissues. Hilleman dubbed the new measles strain “Moraten,” for “More Attenuated Enders.” The new measles vaccine, Attenuvax, was administered without any need for gamma globulin.

Our chronicle continues with the rubella vaccine. Rubella poses its greatest danger to fetuses of non-immune pregnant woman, particularly during the first trimester of pregnancy. In up to 85% of these women, infection will result in a miscarriage or a baby born with severe congenital abnormalities. An outbreak of rubella began in Europe in the spring of 1963, and quickly spread worldwide. In the United States, the 1963 rubella outbreak resulted in the deaths of 11,000 fetuses, and an additional 20,000 others born with birth defects (e.g., deafness, heart disease, cataracts).

Hilleman had been working on a rubella vaccine at the time of the 1963 outbreak. But, he was persuaded to drop his own vaccine and, instead, refine a vaccine (based on a Division of Biologics Standards’ rubella strain) that was at the time too toxic to inoculate into people. By 1969 Hilleman was able to attenuate the DBS strain sufficiently for the vaccine to be approved by the FDA.

Next, and importantly, Hilleman combined the mumps, measles, and rubella vaccines into the single trivalent MMR vaccine, making vaccination and, hence, compliance vastly easier. Thus, MMR was a development that should have been well received by many small children and their mothers, as well as by public health officials.

In 1978 Hilleman found that another rubella vaccine was better than the one in the trivalent vaccine. Its designer, Stanley Plotkin (then at the Wistar Institute), was said to be speechless when asked by Hilleman if his (Plotkin’s) vaccine could be used in the MMR. Merck officials may also have been speechless, considering their loss in revenues. But for Hilleman, it was simply the correct thing to do.

Like Jonas Salk and Albert Sabin before him (3), Maurice Hilleman was never awarded a Nobel Prize. There is no obvious reason for the slight in any of these three instances. In Salk’s case, it may have been because Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. But, Max Theiler received the Nobel Prize for producing a yellow fever vaccine. What’s more, the Nobel committee seemed to equivocate regarding the discovery that might have been involved in that instance. Regardless, the Nobel award to Theiler was the only Nobel Prize ever awarded for a vaccine! [A more complete accounting of the development of Theiler’s yellow fever vaccine can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, now on the blog.]

Sabin had done basic research that perhaps merited a Nobel Prize (3). But, the Nobel committee may have felt uneasy about giving the award to Sabin, without also recognizing Salk. Or, perhaps the continual back-and-forth carping between supporters of Salk and Sabin may have reduced enthusiasm in Stockholm for both of them.

Yet by virtually any measure, Hilleman’s achievements vastly exceeded those of Salk, Sabin, Theiler, and just about everyone else. His basic interferon work alone should have earned him the Prize. Hilleman’s group demonstrated that certain nucleic acids stimulate interferon production in many types of cells, and detailed interferon’s ability to impede or kill many viruses, and correctly predicted its efficacy in the treatment of viral infections (e.g., hepatitis B and C), cancers (e.g., certain leukemias and lymphomas), and chronic diseases (e.g., multiple sclerosis). What’s more, Hilleman developed procedures to mass-produce and purify interferon. And, regarding his unmatched achievements as a vaccinologist, he did more than merely emulate Pasteur’s procedures for developing attenuated viral vaccines. His hepatitis B vaccine was the first subunit vaccine produced in the United States. It was comprised of the hepatitis B surface antigen (HBsAg), which Hilleman purified from the blood of individuals who tended to be infected with hepatitis B virus (e.g., IV drug abusers). Subsequently, to avoid the potential danger of using human blood products in the vaccine, Hilleman developed recombinant yeast cells that produced the HBsAg. And, Hilleman’s meningococcal vaccine was the first vaccine to be based on polysaccharides, rather than on a whole pathogen or its protein subunits.

So, why then was Hilleman bypassed by the Nobel committee? John E. Calfree, in The American, wrote: “As the 80-plus-year-old Hilleman approached death, Offit and other academic scientists lobbied the Nobel committee to award Hilleman the Nobel Prize for Medicine, based partly on his vaccine work and partly on his contributions to the basic science of interferons. The committee made clear that it was not going to award the prize to an industry scientist.” (4) [Paul Offit, referred to here, is the co-developer of the rotavirus vaccine, Rotateq, and a biographer of Hilleman.]

Calfree also notes that Hilleman’s tendency towards self effacement, and his absence from the academic and public spotlight, may also have worked against him. And, unlike Salk, whose name was closely linked to his polio vaccine (3), Hilleman’s name was never associated with any of his nearly forty vaccines. [Yet in the case of Jonas Salk, his public acclaim is generally believed to have hurt him in the eyes of his colleagues and of the Nobel committee.]

Considering the enormity of Hilleman’s contributions, his anonymity was really quite remarkable. As Calfree relates: “In one of the most striking of the dozens of anecdotes told by Offit, Hilleman’s death was announced to a meeting of prominent public health officials, epidemiologists, and clinicians gathered to celebrate the 50th anniversary of the Salk polio vaccine. Not one of them recognized Hilleman’s name!”

With Hilleman’s public anonymity in mind, we conclude our account with the following anecdote. In 1998, a Dr. Andrew Wakefield became a celebrity and hero in the eyes of the public. How this happened, and its consequences are troubling for several reasons, one of which is that it brought undeserved suffering to the self-effacing and benevolent Maurice Hilleman. The Wakefield incident merits, and will have a full-length blog posting of its own. But for now, in 1998 Wakefield authored a report in the prestigious British journal The Lancet, in which he claimed that the MMR vaccine might cause autism in children. The story had a bizarre series of twists and turns, with Wakefield and co-authors eventually issuing a retraction. The immediate cause of the retraction was the disclosure that Wakefield, on behalf of parents of autistic children, had accepted funding to investigate a link between the MMR vaccine and autism. The purpose of the investigation was to determine whether a legal case against the vaccine manufacturer might have merit. In addition to the obvious conflict of interest, Wakefield’s paper had serious technical flaws as well. At any rate, a number of independent studies subsequently demonstrated that there is no causal link between the MMR vaccine and autism. And, in 2010 Wakefield was barred by the British Medical Society from the practice of medicine. But the harm had been done. Hilleman had become the recipient of hate mail and death threats. And, more important to Hilleman I expect, many worried parents, even today, prevent their children from receiving the MMR vaccine (5). Ironically, the very success of the MMR vaccine enabled people to forget just how devastating measles and rubella could be. Maurice Hilleman succumbed to cancer on April 11, 2005.

1. Nature Medicine 11, S2 (2005)
2. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu On the blog.
3. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science On the blog
4. Calfree, J.E., Medicine’s Miracle Man , The American, January 23, 2009
5. Reference 4 contains a somewhat similar tale, in which a 1992 article in Rolling Stone attributed the emergence of HIV to Hillary Koprowski’s polio vaccine. It created a sensation but, as might be expected, there was no evidence to support its premise.

Paralytic poliomyelitis was one of the world’s most feared diseases during the first half of the 20th century. However, the dread of poliovirus ended abruptly with the advent of the poliovirus vaccines in the 1950s. This posting tells the stories of the key players in the race to develop a polio vaccine. In particular, it features the rivalry between Jonas Salk and Albert Sabin, the two main contenders in the pursuit. While their vaccines together have led to the near disappearance of poliovirus worldwide, neither was recognized by the Nobel committee for his achievement. We begin with some background.

Poliovirus has long been especially interesting to me, both as a virologist and personally as well. The reason is that I was a child and young teenager during the annual polio epidemics of the 1940s and early 1950s, and can vividly remember the panic that set in early every summer of the pre-vaccine days, when the first neighbor or schoolmate was stricken. You were kept home from school and couldn’t even play outside. A visit to a hospital in those times was associated with the pitiful sight of young polio victims in the iron lungs that filled the wards, and even the hallways of hospitals back then.

Not even the emergence of AIDS in the early 1980s evoked fear comparable to that brought on by poliomyelitis. Yet despite the dread of poliomyelitis, the disease actually struck many fewer victims than was commonly perceived by the public. The number of poliomyelitis cases in the United States was typically 20,000 to 30,000 per year in the 1940s and 1950s, while influenza still typically kills 40,000 to 50,000 Americans annually. Yet most individuals, then and now seem indifferent to influenza. What’s more, even the 1918 “Spanish Flu” epidemic, which was arguably the most devastating epidemic in human history, did not cause any panic, despite the fact that during the single month of October 1918, it killed 196,000 people in the United States alone! Estimates of the total number killed worldwide by the 1918 Spanish Flu range between 20 million and 50 million.

So, how can we explain the terror brought on by poliomyelitis? It wasn’t simply because poliovirus struck suddenly, without any warning. So did the “Spanish Flu.” Rather, paralytic poliomyelitis mainly struck children, adolescents and young adults. In contrast, influenza mainly threatens the elderly. And, in truth, most parents are far more emotionally invested in their children’s well-being than in that of their parents or themselves. Furthermore, the sight of a child in an iron lung or leg braces (affected legs became atrophied and deformed) was truly heart rending.

The fear evoked by poliomyelitis was permanently ended in the United States and in much of the developed world as well, by the emergence of Salk’s killed polio vaccine in 1955. Sabin’s live attenuated vaccine followed soon after. [Live vaccines generally contain attenuated (weakened) variants of the virulent virus, which can replicate and induce immunity, but which cannot cause disease.] The response of the public to Salk’s vaccine was so great that he was hailed as a “miracle worker.” Nevertheless, and despite the fact that the vaccines created by Salk and Sabin have nearly ridden the world of poliovirus, neither man would ever be recognized by the Nobel committee.

Salk’s public acclaim was resented by his colleagues.

Most virologists of the day strongly favored live vaccines over killed ones, based on the belief that only a live vaccine could induce a level of immunity sufficient to protect against a challenge with live virulent virus. Indeed, the effectiveness of live vaccines had been established much earlier by Jenner’s smallpox vaccine (1798) and Pasteur’s rabies vaccine (1885). Jenner’s smallpox vaccine actually contained live cowpox virus, which was similar enough immunologically to variola to protect against smallpox, while not being able to cause smallpox itself. Pasteur’s rabies vaccine contained live rabies virus that was attenuated by passages through rabbit spinal cords. [Adapting the virus to grow in rabbits attenuated its virulence in humans, while not impairing its ability to induce immunity.] So, bearing in mind the well-established precedence of attenuated vaccines, why did Salk seek to develop a killed vaccine?

In 1941, Thomas Francis, one of the great pioneers of medical virology, working at the University of Michigan, developed a killed influenza vaccine. Providentially, in the same year, Jonas Salk (recently graduated from NYU medical school) came to Francis’ laboratory for postgraduate studies. In Francis’ lab, Salk learned his mentor’s methods for producing his killed influenza vaccine and assisted in its field trials.

Salk’s experience in Francis’ laboratory led him to believe in the potential of a killed poliovirus vaccine. And, Salk learned practical procedures from Francis that would be valuable in his pursuit of that objective. These included the use of formaldehyde to kill the virus, the use of adjuvants to enhance the immunogenicity of the killed vaccine, and protocols for conducting field tests.

In contrast to Salk, Sabin firmly believed that a live attenuated vaccine would be vastly superior to a killed one. And, although Salk won the race to produce an actual vaccine, Sabin had been doing polio research well before the younger Salk emerged on the scene. Indeed, Sabin made several important contributions to the field; some of which are discussed below. For now, we mention that in 1936, Sabin and colleague Peter Olitsky demonstrated that poliovirus could be grown in cultured human embryonic nervous tissue. While this might appear to be a key step towards the development of a vaccine, Sabin and Olitsky feared that nervous tissue might cause encephalitis (inflammation of the brain and spinal cord) when injected into humans.

Albert Sabin, who developed the live polio vaccine.

John Enders, at the Children’s Hospital of Boston, is the next key player in our story. Enders believed that poliovirus should be able to grow in non-nervous tissue, particularly tissue from the alimentary canal, as suggested to him by the amount of the virus that was present in the feces of many patients. So, in 1948, Enders, and colleagues Thomas Weller and Frederick Robbins, succeeded in growing poliovirus in cultured non-nervous tissue, including skin, muscle, and kidney. As a result of Ender’s work, sufficient amounts of poliovirus could now be grown, free from the hazards of nervous tissue, thereby enabling the mass production of a vaccine.

[Aside: Enders, Weller, and Robbins maintained their tissue samples in culture using the roller culture procedure, in which a horizontally positioned bottle is laid on its side and continuously rotated around its cylindrical axis. In comparison to the older process of growing tissues in suspension, the roller culture method enabled the prolonged maintenance of the tissues in an active state and, consequently, the growth of large amounts of virus. For readers who read Renato Dulbecco and the Beginnings of Quantitative Animal Virology (on the blog), note that Dulbecco developed procedures for growing pure cell types as flat adherent monolayer cultures, thereby making possible quantitative plaque assays of animal viruses.]

In 1954, Enders, Weller, and Robbins shared the Nobel Prize in Physiology or Medicine for their contribution described above. What’s more, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research! Ironically, Ender’s true interests actually lay elsewhere; with measles. He would later develop a measles vaccine. [Enders has been referred to as the “Father of modern vaccinology.”]

The next key player of note in our story is not a person but, rather, a foundation; the “National Foundation for Infantile Paralysis,” which led and financed the crusade against polio in the pre-NIH days of the 1950s. The National Foundation was actually an outgrowth of the Georgia Warm Springs Foundation, a charitable organization founded by Franklin D. Roosevelt, himself crippled by polio. However, after Roosevelt became president of the United States, he was too polarizing a figure (particularly after his “court-packing” scheme in 1937) to head up a philanthropic organization. Consequently, in 1938, Roosevelt announced the formation of the nonpartisan National Foundation for Infantile Paralysis.

Photos of Franklin Roosevelt in a wheel chair are rare and were not shown to the public, which was generally unaware that he was paralyzed from the waist down.

[Aside: The National Foundation was initially funded by the contributions of wealthy celebrities who attended Roosevelt’s yearly birthday bashes. At one of these fundraisers, the vaudevillian, Eddie Cantor, jokingly urged the pubic to send dimes directly to the president. And, bearing in mind the fear evoked by polio, the public, perhaps not recognizing the joke, did exactly that, flooding the White House with nearly three million dimes. And so, the slogan “March of Dimes,” for the Foundation’s grass-roots fund-raising campaign, came to be. And, it was not coincidental that a dime (the Roosevelt dime) was issued in 1946 to memorialize the late president.

In 1950, a March of Dimes chapter in Phoenix, Arizona held a “Mother’s March on Polio,” in which volunteers went door-to-door raising money for polio research. People were urged to leave their porch lights on to show that the volunteers would be welcome. The Phoenix initiative soon spread to other locals, and the Mother’s March became a nationwide annual event.]

The role of the National Foundation went beyond merely raising money for research. It also attempted to provide direction to the research, which often placed it at odds with its grantees. This was the case because Harry Weaver, the director of research at the National Foundation, was focused on bringing a vaccine to the public. In contrast, most of the Foundation’s grantees were largely motivated by their desire to understand basic virological issues, such as poliovirus transmission, replication, and dissemination. What’s more, they believed that there was still too much to be known about poliovirus and poliomyelitis before a vaccine might be a realistic possibility.

[Aside: Apropos the sentiment of some poliovirus researchers that there was too much yet to be known before a polio vaccine might be possible, Jenner’s 1798 smallpox vaccine was developed a half century before the germ theory of disease was proposed, and 100 years before the actual discovery of viruses. It was based on the empirical observation that milkmaids seemed to be “resistant” to smallpox; apparently because they had been exposed earlier to cowpox. The initial smallpox vaccine simply contained matter from fresh cowpox lesions on the hands and arms of a milkmaid. It was then serially passed from one individual to another; a practice eventually ended because of the transmission of other diseases. And, Pasteur’s 1885 rabies vaccine too was developed before viruses were recognized as discrete microbial entities.]

Sabin’s objection to the Foundation’s priority of having a vaccine available as quickly as possible was somewhat more personal. Since a killed vaccine should be more straightforward and, therefore, quicker to develop than an attenuated one (see below), Sabin believed that Weaver’s sense of the urgent need for a vaccine would lead him to favor supporting Salk’s killed vaccine over his attenuated one. Moreover, Sabin felt that he was being shunted aside. And, Since Sabin remained firm in his belief in the superiority of a live vaccine; he also felt that Weaver’s main concern of having a vaccine available as quickly as possible, would compromise the efficacy of the vaccine that would be implemented in the end.

[Aside: Back in the Enders laboratory, Thomas Weller and Frederick Robbins wanted to enter the polio vaccine race. But, Enders viewed the project as boring and routine; a view pertinent to the question of why Salk and Sabin were never recognized by the Nobel Committee. Furthermore, Enders didn’t believe that a killed vaccine could ever provide adequate protection against polio, or that a live vaccine would be possible without years more of research.]

Sabin’s worry that a killed vaccine would be faster to develop than an attenuated one was borne out when, in1953, Salk was preparing to carry out a field-test of his killed vaccine. Yet Sabin and other poliovirus researchers remained inclined to move slowly, placing them in opposition to Harry Weaver’s sense of urgency. Moreover, Sabin and the other polio investigators were also piqued at the National Foundation for promoting Salk’s vaccine to the public and, also, for promoting Salk himself as a miracle worker. The Foundation’s reason for publicizing Salk was to stir up public enthusiasm for its fund raising campaigns. And Salk indeed was becoming the symbol of the miracles of medical research to an admiring public.

In fairness to the polio researchers who dissented with the National Foundation’s single minded emphasis on bringing a vaccine to the public, there were valid reasons for believing that the Foundation might be moving too quickly. So, consider the following excerpts from a letter that Sabin wrote to his rival, Salk: “…this is the first time they (the Foundation) have made a public statement based on work which the investigator has not yet completed or had the opportunity to present…in a scientific journal…Please don’t let them push you to do anything prematurely or to make liters of stuff for Harry Weaver’s field tests until things have been carefully sorted out, assayed, etc., so that you know what the score is before anything is done on a public scale.”

While Sabin’s advice to Salk seems eminently sensible, Sabin had never before shown any inclination to look out for Salk’s interests. So, might Sabin be sending a non-too-subtle warning to Salk that he could either play by the traditions of the scientific community, or face the consequences of playing to the interests of the Foundation? For his part, Salk was well aware of what was happening and he was indeed embarrassed by the adulation of the press; correctly sensing that it was compromising his standing with his colleagues.

[Aside: The media, in the person of the legendary broadcaster, Edward R. Murrow, provided Salk with a notable and very satisfying moment in the public spotlight. During an April, 1955 interview on the CBS television show See it Now, Murrow asked Salk: “Who owns the patent on this vaccine?” To which, Salk replied: “Well, the people, I would say. There is no patent. Could you patent the sun?”

While Salk’s answer to Murrow endeared him even more to the public, some colleagues questioned whether it might have been disingenuous. Both the University of Pittsburgh, where Salk carried out his work, and the National Foundation, which financed it, indeed had been looking into the possibility of patenting Salk’s vaccine. But, when patent attorneys sought to determine if there was a basis for a patent, Salk readily acknowledged that his vaccine was, for the most part, based on tried and true procedures developed by others.

In point of fact, Salk’s critics held him in low esteem largely because there was little about his vaccine that was innovative. Indeed, Sabin once quipped: “You could go into the kitchen and do what he (Salk) did.” But in fairness to Salk, he never claimed that his vaccine was unique. Instead, in the face of much skepticism, his point had always been that a killed vaccine could protect against polio. He persevered and he was right.

Note that Sabin too gave his vaccine to the world gratis.]

By 1954, field tests of Salk’s vaccine went ahead on a massive scale, involving nearly 1.5 million schoolchildren nationwide. The tests were overseen by Thomas Rivers, an eminent virologist who, at the time, was Director of the Rockefeller Institute. Like most virologists, Rivers favored a live vaccine as the ultimate solution to polio. Nevertheless, he believed that the world couldn’t wait ten or more years for an ideal vaccine, when a satisfactory one might be available at present.

With 57,879 cases of poliomyelitis in the United States in 1952, the peak year of the epidemic, Harry Weaver’s sense of the urgent need for a vaccine was widely shared by the public. Unsurprisingly then, the public eagerly supported the 1954 field test of Salk’s vaccine, as indicated by the fact that 95% of the children in the test received all three required vaccinations. [Killed vaccines require multiple doses. That is so because the first dose only primes the immune system. The second or third dose then induces the primed immune system to produce protective antibodies against the virus. Inoculation with a live vaccine resembles a natural infection and, consequently, a single dose is sufficient to induce immunity.]

The field test of Salk’s vaccine was unprecedented in its size. What’s more, it was supported entirely by the National Foundation, which strenuously opposed outside interference from the federal government. In actuality, the Foundation considered federal funding for polio research to be a “Communistic, un-American…scheme.”

[Aside: President Dwight Eisenhower, a Republican and a fiscal conservative, also believed that the federal government had no proper a role in health care. Consequently, Eisenhower took little interest in his Department of Health, Education, and Welfare (HEW). What’s more, Eisenhower’s Secretary of HEW, Oveta Culp Hobby, was even more conservative in that regard than Eisenhower himself. In 1955, after the field trials showed the Salk vaccine to be a success, and with the public clamoring for it, there were insufficient amounts of the vaccine available to meet the public’s demands. Thus, even some Republicans were stunned to learn that the Eisenhower administration had taken no actions whatsoever to watch over production of the vaccine or its distribution, believing that this was in the province of the drug companies. When pressed on this, Mrs. Hobby responded: “I think no one could have foreseen the public demand.”

Not surprisingly, American drug companies lobbied intensely to keep vaccine production under their own control. A different scenario played out in Canada, where the government viewed polio as a national crisis, and took control of its vaccination program, with overwhelming public support.]

All did not go well for Salk and his vaccine after the successful 1954 field tests. In April 1955, more than 200,000 children were inoculated with a stock of improperly inactivated vaccine made by Cutter Laboratories; one of the five companies that produced the vaccine in 1955. [The others were Eli Lilly, Parke-Davis, Wyeth, and Pitman-Moore.] The Cutter vaccine caused 40,000 cases of abortive poliomyelitis (a form of the disease that does not involve the central nervous system), and 56 cases of paralytic poliomyelitis; 5 of which were fatal. What’s more, some of the children inoculated with the Cutter vaccine transmitted the vaccine virus to others, resulting in 113 more cases of paralytic poliomyelitis and 5 fatalities.

A congressional investigation blamed the “Cutter incident” on the NIH Laboratory of Biologics Control, for insufficiently scrutinizing the vaccine producers. In point of fact, the NIH did little testing on its own. Instead, it mainly relied on reports from the National Foundation, whose agenda was to proceed with the vaccinations. Yet the NIH did have an early, in-house warning of potential problems with the Cutter vaccine, which it failed to act on. Bernice Eddy, a staff microbiologist at the NIH, reported to her superiors that the Cutter vaccine caused paralysis when inoculated into monkeys. However, no action was taken in response to Eddy’s warning. [In 1959, Eddy discovered simian virus 40 (SV40) in monkey kidney tissue that was used for vaccine production. By that time, live SV40 had unknowingly been injected into hundreds of millions of people worldwide; perhaps the subject of a future blog posting.]

Salk was exonerated of any fault in the Cutter incident. Moreover, after that episode, not a single case of polio in the United States would be attributed to Salk’s vaccine. Nevertheless, while Salk’s killed vaccine was perfectly safe when properly prepared, the Cutter incident led to the perception that it was unsafe. Consequently, Salk’s killed vaccine was eventually replaced by Sabin’s live attenuated one. Ironically, as we will see, the perception that Salk’s vaccine was dangerous led to its replacement by a more dangerous one.

Sabin’s work on his live polio vaccine began in 1951 and, like Salk; he was supported by the National Foundation. Sabin’s task was more difficult than Salk’s because it is more straightforward to kill poliovirus, than it is to attenuate it. [The attenuated virus must be able to replicate in the digestive tract and induce immunity, yet be unable to damage the nervous system.] But Sabin persisted, sustained by his conviction that a live vaccine would invoke stronger, longer-lasting immunity than a killed vaccine. Sabin attenuated his vaccine by successive passages through monkey tissue, until the live virus could no longer cause paralysis when inoculated directly into chimpanzee spinal cords.

[Aside: At this early date, live-vaccine-proponents could not have known that only a live vaccine could activate T-cell mediated immunity, which is generally necessary to clear a virus infection. Instead, their preference for live vaccines was based on the simpler, but correct notion that inoculation with a live vaccine would more closely approximate a natural infection. Also, since the vaccine virus is alive, vaccinated individuals might transmit it to unvaccinated ones, thereby inducing immunity in the latter as well. On the other hand, the attenuated vaccine poses a deadly threat to individuals with impaired immune systems, such as AIDS patients and individuals on immunosuppressive regimens following organ transplants.]

In 1954, a successful small-scale test of Sabin’s vaccine was carried out, which involved thirty adult human prisoners at a federal detention facility. The promising outcome of this test warranted a larger field-trial of Sabin’s vaccine. But, several obstacles stood in the way. First, the National Foundation was not inclined to support another massive field trial, now that Salk’s vaccine was already in use. Second, the Foundation was still reeling from the Cutter incident, and had no inclination to be caught up in another such debacle. Third, it would be virtually impossible to conduct the trials in the United States, since millions of American children had already been inoculated with Salk’s vaccine. The ensuing course of events was rather remarkable.

By 1956, poliomyelitis had become a serious public health crisis in the former Soviet Union. Consequently, a delegation of Russian scientists came to the United States to meet with Salk and consult with him on how to produce his vaccine. However, the Russians were disposed to meet with other polio researchers as well. Thus, Sabin seized this opportunity to invite the Russians to visit his laboratory at the University of Cincinnati, where he was able to tout his live vaccine to them. Sabin’s pitch was apparently effective, as he secured an invitation from the Russians to visit the Soviet Union, where he spent a month, further hyping his vaccine.

[Aside: While Sabin was in Russia, the Russians requested from him a sample of his live vaccine. So, when Sabin returned to the United States, he sought permission from the State Department to send the Russians the samples they requested. The State Department approved the request; but it did so over objections from the Defense Department, which was concerned that the vaccine virus might have “biological warfare applicability.”]

With the incidence of poliomyelitis on the rise in the Soviet Union, the Soviet Health Ministry needed to quickly decide which vaccine to adopt; Salk’s or Sabin’s. The Russians were already producing the Salk vaccine, but were unable to consistently maintain its efficacy from one batch to another. So, the Soviets invited Salk to visit Russia, so that he might help them to solve the problems they were having producing his vaccine.

Salk then made a decision that he would long regret. Because of pressure from his wife to spend more time with his family, Salk turned down the Russian invitation. The upshot was that the Russians turned instead to Sabin. In 1959 they vaccinated 10 million children with vaccine strains sent to them by Sabin. Soviet results with the Sabin vaccine were so promising that the Soviet Health Ministry decided to then use it to vaccinate everyone under 20 years of age. A total of seventy-seven million Soviet citizens were vaccinated with Sabin’s vaccine, vastly exceeding the number vaccinated during field trials of the Salk vaccine in the United States.

The U.S. Public Health Service did not endorse the Sabin vaccine for use in the United States until 1961. By then, the Salk vaccine had virtually eliminated polio from the country. Nevertheless, Sabin’s vaccine supplanted Salk’s in the United States and in much of the rest of the world as well.

Yet all did not go well with Sabin’s vaccine either. As noted above, after the Cutter incident, there were no cases of poliomyelitis in the United States that could be attributed to Salk’s vaccine. In contrast, Sabin’s vaccine caused about a dozen polio cases per year, a frequency of about one case per million vaccinated individuals. At least some of these cases resulted from the ability of the attenuated virus to revert to a more virulent form. What’s more, reverting viruses posed a threat to non-vaccinated individuals in the population. For instance, in 2000/2001, there were 21 confirmed cases of poliomyelitis in the Dominican Republic and Haiti, which were traced to a single dose of the Sabin vaccine that was administered during the preceding year. [As noted in an above Aside, since the Sabin vaccine is alive, vaccinated individuals might transmit the vaccine virus to unvaccinated individuals.]

In actual fact, the few cases of poliomyelitis that now occur in the West are vaccine-related, resulting from the rare reversions of Sabin’s vaccine. Ironically, the Sabin vaccine, which played a crucial role in the near eradication of polio from the world, had become an obstacle to the complete eradication of the virus. In 2000, the U.S. Centers for Disease Control (CDC) recommended the complete return to the Salk vaccine in the United States. However, the Sabin vaccine would continue to be used in much of the developing world.

[Aside: Several polio hotspots remain in the world. Three major ones are Pakistan, Afghanistan, and Nigeria. Recent outbreaks have also occurred in Syria and Somalia. In each of these instances, social and political climates make it difficult to carry out eradication campaigns.

As recently as March 2014, militants attacked a polio vaccination team in northwest Pakistan, detonating a roadside bomb and then opening fire on their convoy, killing 12 of their security team, and wounding dozens more. Some Pakistani religious leaders denounced the vaccination campaign in Pakistan as a cover for spying or as a plot to sterilize Muslim children.

In the developed world there is a very different problem. Ironically, the great success with which the polio vaccines eradicated the virus in the West has created conditions there in which poliomyelitis might make a most unwelcome return. That has come about because too many parents in the developed world now view polio as ancient history, and have become complacent about having their children vaccinated. What’s more, some parents are heeding unsubstantiated warnings that the risks of vaccines are greater than the risks of the viruses. Consequently, the frequency of vaccinated individuals in the West is declining to the point where the West may be susceptible to outbreaks sparked by imported cases. These issues will be discussed at length in a subsequent posting.]

We turn now to an issue raised at the outset of this posting; neither Salk nor Sabin was recognized by the Nobel Committee for his contribution. That is so, despite the fact that their individual efforts, taken together, have virtually eliminated polio from the world.

Max Theiler, at the Rockefeller Institute, is relevant regarding the Nobel issue, and for several other reasons as well. First, Theiler took an early interest in Sabin’s career during Sabin’s years at the Rockefeller (1935 to 1939). Second, during those years Theiler was working on a live attenuated vaccine for yellow fever. Like most virologists of the day, Theiler believed that only a live vaccine could provoke significant long-lasting immunity. And, Theiler’s thinking on this matter likely influenced Sabin’s later approach to a polio vaccine. Thirdly, and important in the current context, in 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for his yellow fever vaccine. Fourth, Theiler’s Nobel Prize was the only one ever awarded for the development of a virus vaccine!

Why was Theiler’s Nobel award the only one ever given for the development of a virus vaccine? In addition, recall that John Enders, Thomas Weller, and Frederick Robbins shared the 1954 Nobel Prize for Physiology or Medicine, for demonstrating that poliovirus could be propagated in non-nervous tissue. Moreover, the Nobel Prize shared by Enders, Weller, and Robbins was the only one ever given in recognition of polio research! Why weren’t Salk and Sabin recognized as well? Didn’t they also contribute substantially “to the benefit of mankind;” a standard for the award, as specified by Alfred Nobel?

Apropos these questions, it may be relevant that Alfred Nobel also specified that the prize for physiology or medicine should recognize a “discovery” per se. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. In contrast, the discovery of Enders, Weller, and Robbins, refuted the previously held belief that poliovirus could be grown only in nervous tissue; a breakthrough that paved the way to the vaccines.

But then, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur developed an attenuated Rabies vaccine in 1885? And, what of Jenner’s earlier 1798 smallpox vaccine, comprised of live cowpox virus?

To the above points, Sven Gard, at the Karolinska Institute, and a member of the Nobel committee for Physiology or Medicine, wrote the following in his evaluation of Theiler’s prior 1948 Nobel nomination: “Theiler can not be said to have been pioneering. He has not enriched the field of virus research with any new and epoch-making methods or presented principally new solutions to the problems, but he has shown an exceptional capacity to grasp the essentials of the observations, his own and others, and with safe intuition follow the path that led to the goal.”

Despite the seeming inconsistency between Gard’s comments and Nobel’s instruction that the prize be awarded for a discovery, Gard nonetheless concluded that Theiler’s contributions indeed merited the Nobel award. [Incidentally, Theiler’s 1948 Nobel nomination was a detailed six-page-long document, written and submitted on his behalf by Albert Sabin!]

To the same point, Hilding Bergstrand, also at the Karolinska Institutet, and chairman of the Nobel Committee for Physiology and Medicine, said the following during his otherwise laudatory speech honoring Theiler at the 1951 Nobel Prize ceremony: “The significance of Max Theiler’s discovery must be considered to be very great from the practical point of view, as effective protection against yellow fever is one condition for the development of the tropical regions—an important problem in an overpopulated world. Dr. Theiler’s discovery does not imply anything fundamentally new, for the idea of inoculation against a disease by the use of a variant of the etiological agent which, though harmless, produces immunity, is more than 150 years old.”

Even Theiler himself agreed that he had not done anything fundamentally new. But then, what might Bergstrand have had in mind when referring to Theiler’s discovery? Perhaps it was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryos, which were devoid of nervous systems, generated viable, non-neurotropic attenuated yellow fever virus. If so, then did that discovery fulfill the condition for the Nobel award, as specified by Alfred Nobel? And, if that is the case, then might this discovery have been what makes Theiler’s contribution more worthy than those of Salk and Sabin in the eyes of the Nobel committee? [A more detailed account of Max Theiler’s yellow fever vaccine, particularly with regard to the “discovery” noted here, can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiller, now on the blog.]

The seemingly trivial distinction between the worthiness of Theiler’s contribution from that of Salk and Sabin, suggests that we may need to look elsewhere for answers to why Salk and Sabin were bypassed by the Nobel committee. One reason suggested in the case of Salk is that in the elitist world of big-time science, he had never spent time at a prestigious Research institution like the Rockefeller. Yet he did carry out postgraduate studies in association with the eminent Thomas Francis. So perhaps he was passed over by the Nobel committee because it did not see anything innovative about his vaccine. Or, perhaps it was because he allowed himself to be promoted as a celebrity by the March of Dimes, thereby causing resentment among his colleagues.

But, how then might we explain the case of Sabin? Sabin had not been used by the National Foundation to promote its fund-raising. And, he had done research at the Rockefeller Institute. Moreover, Sabin made seminal contributions to the poliovirus field before and after beginning his vaccine work. As noted above, Sabin and Peter Olitsky demonstrated that poliovirus could be grown in cultured human embryonic nervous tissue. Moreover, Sabin provided experimental evidence that the poliovirus port of entry is the digestive tract, rather than the respiratory tract, as was previously thought. And, Sabin established that the incidence of poliomyelitis tended to be highest in urban populations which had the highest standards of sanitation.

[Aside: Sabin’s finding, that the poliovirus route of entry is via the alimentary tract, validated the premise that poliomyelitis might be prevented by a live oral vaccine. In contrast, Salk’s killed vaccine needed to be injected. An advantage of a vaccine being administered by the oral route, particularly in developing countries, is that trained medical personnel are not required for its administration. On the other hand, the killed vaccine is safer. The few cases of poliomyelitis that now occur in the West are vaccine-related, resulting from rare reversions to virulence of the attenuated virus.]

[Aside: Why was the incidence of poliomyelitis highest in urban populations that had the highest standards of hygiene? Polio infection tends to be milder in the very young, perhaps because they are partially protected by maternal antibodies. But, in areas with high standards of hygiene, infection tends to occur later in life, when maternal antibodies have waned, and the infection can then be more severe.

Before this was appreciated, poliomyelitis was thought to originate in the slums and tenements of cities, and then spread to the cleaner middle-class neighborhoods. Thus, during polio outbreaks in New York City, there were instances when slums and tenements were quarantined, and city dwellers fled to the suburbs, all to no avail.]

Were Sabin’s discoveries noted above, taken together with his vaccine, worthy of a Nobel Prize? In any case, Sabin indeed had been nominated for the Nobel award by numerous colleagues, including Enders. So, why was Sabin never awarded the Nobel Prize? Perhaps the Nobel committee could not recognize Sabin without also recognizing Salk, which it may have been reluctant to do for reasons noted above. Or, as has been suggested, the continual back-and-forth carping between supporters of Salk and Sabin may ultimately have diminished enthusiasm in Stockholm for both of them.

Salk (in 1956) and Sabin (in 1965) each received the prestigious Lasker Award for Clinical Research (often seen as a prelude to the Nobel) and, earlier, in 1951, Sabin was elected to the U.S. National Academy of Sciences. In contrast, Salk was the only prominent polio researcher not elected to the Academy. And regarding the Nobel Prize, Salk once joked that he didn’t need it, since most people thought he had already won it.

In 1963 Salk founded the prestigious Salk Institute for Biological Studies in La Jolla, California. Francis Crick (1), Renato Dulbecco (2), and Leo Szilard (3), each of whom is featured elsewhere on the blog, were among the eminent scientists recruited by Salk to the La Jolla campus. Bearing in mind Salk’s alienation from other medical researchers of the day, we might enjoy his remark “I couldn’t possibly have become a member of this institute if I hadn’t founded it myself.” Jonas Salk died of congestive heart failure in 1995 at the age of 80. He remains one of the most venerated medical scientists ever.

Salk Institute for Biological Studies

[Aside: Salk married Dora Lindsay in 1939, right after he graduated from NYU medical school. But, the marriage eventually fell apart, and the couple divorced in 1968.

In 1970, Salk married the artist Francois Gilot, who had been the mistress of Pablo Picasso for nearly ten years and with whom she had two children. Salk and Gilot met in 1969, at the home of a mutual friend in Los Angeles. They remained married until Salk’s death in 1995.

The following is from an April 27, 2012 article in Vogue by Dodie Kazanjian, entitled Life after Picasso: Francois Gilot.

“On a trip to Los Angeles in 1969, a friend introduced her to Jonas Salk. She had no interest in meeting him—she thought scientists were boring. But soon afterward, he came to New York and invited her to have tea at Rumplemayer’s. ‘He didn’t have tea; he ordered pistachio and tangerine ice cream,’ she recalls. ‘I thought, Well, a scientist who orders pistachio and tangerine ice cream at five o’clock in the afternoon is not like everybody else!’ He pursued her to Paris and a few months later asked her to marry him. She balked. “I said, ‘I just don’t need to be married,’ and he said, ‘In my position, I cannot not be married.’ He gave me two pieces of paper and told me to write down the reasons why I didn’t want to get married.” She complied. Her list included: ‘I can’t live more than six months with one person’; ‘I have my own children’; ‘I have my career as a painter and have to go here and there’; ‘I’m not always in the mood to talk. Et cetera, et cetera, et cetera.’

Salk looked at the list and said he found it ‘quite congenial.’ They were married in 1970 and were together until he died in 1995. ‘It worked very well,’ she says, because after all we got along very well.’”]

Albert Sabin became president of the prestigious Weizmann Institute of Science in Israel, but stepped down in November 1972 for health reasons. He passed away in 1993 at the age of 86. Unlike in the case of Salk, and despite the fact that he never was awarded the Nobel Prize, Sabin’s standing among his colleagues always remained high.

Before concluding, we note two other important contenders in the quest for a polio vaccine. The first of these was Isabel Morgan, the daughter of the great geneticist, Thomas Hunt Morgan. Isabel Morgan nearly produced a killed polio vaccine before Salk succeeded in doing so. Working at Johns Hopkins, she generated formalin-inactivated poliovirus preparations that indeed protected monkeys against intracerebral injections of live poliovirus. However, Morgan gave up her research in 1949 to marry and raise a family. At that time, Salk had barely begun his work. But, if Morgan had remained in the race, Salk may yet have beaten her to the finish line, since she was reluctant to test her vaccine on human subjects.

Hilary Koprowski was the other noteworthy contender in the race to a polio vaccine. Koprowski was a Polish Jew who immigrated to Brazil in 1939, after Germany invaded Poland. He later came to the United States, where, in 1945, he was hired by Lederle Laboratories to work on a project to develop a live polio vaccine. Koprowski’s foray into polio had a few interesting happenings. Moreover, he went on to have a renowned career as a virologist. Thus, we discuss him in a bit more detail.

[Aside: Salk and Sabin also were Jewish. And Sabin too was born in Poland. In 1921 he immigrated with his family to the United States, at least partly to escape persecution of Jews in his birth-land.]

Koprowski began his work at Lederle before John Enders developed methods for growing poliovirus in monkey kidney cell cultures. Consequently, Koprowski attenuated his live vaccine by passaging it in mouse brains in vivo. In 1950, several years before Sabin’s vaccine was ready for testing, Koprowski found that his vaccine indeed protected chimpanzees from challenge with virulent poliovirus. Koprowski then tested his live vaccine in humans; first on himself, and then on 19 children at a New York State home for “feeble minded” children.

Koprowski was still an unknown figure in the scientific community when he made the first public presentation his test findings. This happened at a 1951 National Foundation roundtable that was attended by the major polio researchers of the day, including Salk and Sabin. The conferees were aghast upon hearing that Koprowski had actually tested his live vaccine, grown in animal nerve tissue, on children. Koprowski’s response was simply that someone had to take that step. Also, it didn’t help Koprowski’s standing with his academic colleagues that he was employed by Lederle. In those pre-biotech days, he was looked down on as a “commercial scientist.”

Human testing was of course a necessary step in the development of this or any human vaccine. What’s more, using cognitively disabled children as test subjects was a common practice back then. So, the actual concern of Koprowski’s colleagues was that he inoculated human subjects with a vaccine that was grown in animal brains. Koprowski also may have been treading on shaky legal ground, since it is not clear whether he ever obtained consent from the children’s parents.

[Aside: The only guidelines for such tests back then were the so-called Nuremburg Code of 1947, which was formulated in response to Nazi “medical” experiments. Informed consent was one of the Nuremburg guidelines, which, in the case of children, meant consent from a parent or guardian. Note that federal approval was not required to test vaccines or drugs in those days.]

Irrespective of whatever uproar Koprowski caused by testing his vaccine on helpless institutionalized children, he indeed had a live polio vaccine in 1949; several years before Salk and Sabin brought out their vaccines. However, Koprowski’s vaccine began its demise soon afterwards. A small field trial in Belfast showed that the attenuated virus could revert to a virulent form after inoculation into humans. But, bearing in mind that there was not yet any alternative to his vaccine, Koprowski firmly believed that the greater risks of natural poliovirus infections justified its use.

The fate of Koprowski’s vaccine was sealed in 1960, when the U.S. Surgeon General approved the Sabin vaccine for trial manufacture in the United States, while rejecting Koprowski’s vaccine on safety grounds. Tests showed that Sabin’s vaccine was the less neurovirulent of the two vaccines in monkeys. Sabin had carefully tested plaque-isolated clones of his attenuated viral populations for neurovirulence in monkeys, and he then assembled his vaccine from the least neurovirulent of these clones. Moreover, by this time millions of children in the Soviet Union had had been successfully immunized with the Sabin vaccine.

Koprowski left Lederle Laboratories in 1957 after clashing with its management. After that, he became Director of the Wister Institute in Philadelphia. He then transformed the then moribund Wistar into a first class research organization.

The relationship between Koprowski and Sabin was quite adversarial at the time their vaccines were in competition, but they later became friends. In 1976, Koprowski was elected to the U.S. National Academy of Sciences, an honor shared with Sabin, bit never afforded to Salk.

Here is one last bit on Koprowski. Recall that early lots of the Salk and the Sabin vaccines unknowingly contained live SV40, which had been injected into hundreds millions of people worldwide. While the unknown presence of a live tumor virus in a vaccine must be one of a vaccinologist’s worst nightmares, this finding did not attract the attention of the public. In contrast, a 1992 article in Rolling Stone, which attributed the emergence of HIV to Koprowski’s polio vaccine, created a sensation. The premise of the article was that Koprowski’s vaccine was produced in chimpanzee cells that were contaminated with simian immunodeficiency virus (SIV), which then mutated into HIV when inoculated into humans. As might be expected, there was no evidence to support that premise. Indeed, PCR analysis could not detect SIV or HIV in the supposedly contaminated vaccine lots, and records from Koprowski’s laboratory showed that his vaccine was never grown in chimpanzee cells. So, faced with the possibility of a lawsuit, Rolling Stone issued a retraction.

Readers, who enjoyed the above account of the rivalry between Jonas Salk and Albert Sabin, may also enjoy the account of the rivalry between Robert Gallo and Luc Montagnier in Who Discovered HIV? More on the same topic can be found in How the Human Immunodeficiency Virus (HIV) Got its Name. For a very different kind of rivalry, that between Howard Temin and David Baltimore, see Howard Temin: In From the Cold.

1. Howard Temin: “In from the Cold” On the blog.

2. Renato Dulbecco and the Beginnings of Quantitative Animal Virology On the blog.

Blogs I Follow

Welcome!

I am now a retired professor emeritus of Microbiology at the University of Massachusetts. Teaching virology has been a most rewarding aspect of my career. I especially enjoyed enlivening my lectures with a variety of relevant anecdotes.

Virology Textbook

Based on my experiences teaching virology for more than 35 years, I wrote Virology: Molecular Biology and Pathogenesis (ASM Press; 2010). For info on adopting or buying this textbook, please visit the publisher site: http://www.asmscience.org/content/book/10.1128/9781555814533